Proteins and nucleic acids useful in vaccines targeting Staphylococcus aureus

ABSTRACT

Disclosed are novel immunogenic proteins derived from  Staphylococcus aureus , as well as methods for their use in conferring protective immunity against  S. aureus  infections. Also disclosed are nucleic acids encoding the proteins and methods of use of these nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

In accordance with 37 C.F.R. 1.76, a claim of priority is included in anApplication Data Sheet filed concurrently herewith. Accordingly, thepresent invention claims priority as a divisional of U.S. patentapplication Ser. No. 14/110,475, filed Nov. 25, 2013, which is a §371national stage entry of International Application No. PCT/EP2012/056069,filed Apr. 3, 2012, which claims priority to Danish Patent ApplicationNo. PA 2011 70167, filed Apr. 8, 2011 and U.S. Provisional ApplicationNo. 61/473,376, filed Apr. 8, 2011, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of antimicrobial prophylaxisand therapy. In particular the present invention relates to novelproteins and polynucleotides derived from Staphylococcus aureus. Theinvention further relates to vectors comprising the polynucleotides,transformed host organisms expressing the polynucleotides, antibodies(mono- or polyclonal) specific for the polypeptides as well asdiagnostic, prophylactic and therapeutic uses and methods. Finally, alsomethods of preparation are part of the invention.

BACKGROUND OF THE INVENTION

Bacterial infections are in most instances successfully treated byadministration of antibiotics to patients in need thereof. However, dueto careless or thoughtless use of powerful antibiotics, manypathological germs become resistant against antibiotics over time. Onethreatening example is Staphyloccocus aureus. In particular in hospitalsthis bacterium is of relevance. So-called Methicillin Resistant S.Aureus (MRSA) strains jeopardize patient's survival in hospitals, inparticular after surgery.

Vaccination is considered to be a very effective method of preventinginfectious diseases in human and veterinary health care. Vaccination isthe administration of immunogenically effective amounts of antigenicmaterial (the vaccine) to produce immunity to a disease/disease-causingpathogenic agent. Vaccines have contributed to the eradication ofsmallpox, the near eradication of polio, and the control of a variety ofdiseases, including rubella, measles, mumps, chickenpox, typhoid fever.

Before “the genomic era”, vaccines were based on killed or liveattenuated, microorganisms, or parts purified from them. Subunitvaccines are considered as a modern upgrade of these types of vaccine,as the subunit vaccines contain one or more protective antigens, whichare more or less the weak spot of the pathogen. Hence, in order todevelop subunit vaccines, it is critical to identify the proteins, whichare important for inducing protection and to eliminate others.

An antigen is said to be protective if it is able to induce protectionfrom subsequent challenge by a disease-causing infectious agent in anappropriate animal model following immunization.

The empirical approach to subunit vaccine development, which includesseveral steps, begins with pathogen cultivation, followed bypurification into components, and then testing of antigens forprotection. Apart from being time and labour consuming, this approachhas several limitations that can lead to failure. It is not possible todevelop vaccines using this approach for microorganisms, which cannoteasily be cultured and only allows for the identification of theantigens, which can be obtained in sufficient quantities. The empiricalapproach has a tendency to focus on the most abundant proteins, which insome cases are not immuno-protective. In other cases, the antigenexpressed during in vivo infection is not expressed during in vitrocultivation. Furthermore, antigen discovery by use of the empiricalapproach demands an extreme amount of proteins in order to discover theprotective antigens, which are like finding needles in the haystack.This renders it a very expensive approach, and it limits the vaccinedevelopment around diseases, which is caused by pathogens with a largegenome or disease areas, which perform badly in a cost-effectiveperspective.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide S. aureusderived antigenic polypeptides that may serve as constituents invaccines against S. aureus infections and in diagnosis of S. aureusinfections. It is also an object to provide nucleic acids, vectors,transformed cells, vaccine compositions, and other useful means formolecular cloning as well as for therapy and diagnosis with relevancefor S. aureus.

SUMMARY OF THE INVENTION

It has been found by the present inventor(s) that S. aureus, inparticular drug resistant S. aureus, expresses a number of hithertounknown surface exposed proteins which are candidates as vaccine targetsas well as candidates as immunizing agents for preparation of antibodiesthat target S. aureus.

So, in a first aspect the present invention relates to a polypeptidecomprising

-   a) an amino acid sequence selected from the group consisting of any    one of SEQ ID NOs: 1-19, or-   b) an amino acid sequence consisting of at least 5 contiguous amino    acid residues from any one of SEQ ID NOs: 1-19, or-   c) an amino acid sequence having a sequence identity of at least 60%    with the amino acid sequence of a),-   d) an amino acid sequence having a sequence identity of at least 60%    with the amino acid sequence of b), or-   e) an assembly of amino acids derived from any one of SEQ ID NOs:    1-19 which has essentially the same 3D conformation as in the    protein from which said assembly is derived so as to constitute a    B-cell epitope,-   said polypeptide being antigenic in a mammal.

In another aspect, the invention relates to an isolated nucleic acidfragment, which comprises

-   i) a nucleotide sequence encoding a polypeptide of the invention, or-   ii) a nucleotide sequence consisting of any one of SEQ ID NOs:    20-57.-   iii) a nucleotide sequence consisting of at least 10 consecutive    nucleotides in any one of SEQ ID NOs: 20-57,-   iv) a nucleotide sequence having a sequence identity of at least 60%    with the nucleotide sequence in i) or ii),-   v) a nucleotide sequence having a sequence identity of at least 60%    with the nucleotide sequence in iii),-   vi) a nucleotide sequence complementary to the nucleotide sequence    in i)-v), or-   vii) a nucleotide sequence which hybridizes under stringent    conditions with the nucleotide sequence in i)-vi).

In a third aspect, the invention relates to a vector comprising thenucleic acid of the invention, such as a cloning vector or an expressionvector.

In fourth aspect, the invention relates to a cell which is transformedso as to carry the vector of the invention.

In a fifth aspect, the invention relates to a pharmaceutical compositioncomprising a polypeptide of the invention, a nucleic acid fragment ofthe invention, a vector of the invention, or a transformed cell of theinvention, and a pharmaceutically acceptable carrier, vehicle ordiluent.

In a sixth aspect, the invention relates to a method for inducingimmunity in an animal by administering at least once an immunogenicallyeffective amount of a polypeptide of the invention, a nucleic acidfragment of the invention, a vector of the invention, a transformed cellof the invention, or a pharmaceutical composition of the fifth aspect ofthe invention so as to induce adaptive immunity against S. aureus in theanimal.

In a seventh and eighth aspect, the invention relates to 1) a polyclonalantibody in which the antibodies specifically bind to at least onepolypeptide of the invention, and which is essentially free fromantibodies binding specifically to other S. aureus polypeptides, and to2) an isolated monoclonal antibody or antibody analogue which bindsspecifically to a polypeptide of the invention. In a related ninthaspect, the invention relates to a pharmaceutical composition comprisingsuch a polyclonal or monoclonal antibody and a pharmaceuticallyacceptable carrier, vehicle or diluent.

In a 10^(th) aspect, the invention relates to a method for prophylaxis,treatment or amelioration of infection with S. aureus, in particularinfection with multi-resistant S. aureus, comprising administering atherapeutically effective amount of an antibody of the 7^(th) or 8^(th)aspect of the invention or a pharmaceutical composition of the eighthaspect to an individual in need thereof.

In an 11^(th) aspect, the invention relates to a method for determining,quantitatively or qualitatively, the presence of S. aureus, inparticular the presence of multi-resistant S. aureus, in a sample, themethod comprising contacting the sample with an antibody of aspects 8 or9 of the invention and detecting the presence of antibody bound tomaterial in the sample.

In an 12^(th) aspect of the invention is provided a method fordetermining, quantitatively or qualitatively, the presence of antibodiesspecific for S. aureus, in particular the presence of antibodiesspecific for multi-resistant S. aureus, in a sample, the methodcomprising contacting the sample with a polypeptide of the invention anddetecting the presence of antibody that specifically bind saidpolypeptide.

In a 13^(th) aspect, the invention relates to a method for determining,quantitatively or qualitatively, the presence of a nucleic acidcharacteristic of S. aureus, in particular the presence of a nucleicacid characteristic of multi-resistant S. aureus, in a sample, themethod comprising contacting the sample with a nucleic acid fragment ofthe invention and detecting the presence of nucleic acid in the samplethat hybridizes to said nucleic acid fragment.

In a 14^(th) aspect, the invention relates to a method for thepreparation of the polypeptide of the invention, comprising

-   -   culturing a transformed cell of the present invention, which is        capable of expressing the nucleic acid of the invention, under        conditions that facilitate that the transformed cell expresses        the nucleic acid fragment of the invention, which encodes a        polypeptide of the invention, and subsequently recovering said        polypeptide, or    -   preparing said polypeptide by means of solid or liquid phase        peptide synthesis.

In a 15^(th) aspect, the invention relates to a method for determiningwhether a substance, such as an antibody, is potentially useful fortreating infection with S. aureus, the method comprising contacting thepolypeptide of the invention with the substance and subsequentlyestablishing whether the substance has at least one of the followingcharacteristics:

-   1) the ability to bind specifically to said polypeptide,-   2) the ability to compete with said polypeptide for specific binding    to a ligand/receptor, and-   3) the ability to specifically inactivate said polypeptide.

Finally, in a 16^(th) aspect, the invention relates to a method fordetermining whether a substance, such as a nucleic acid, is potentiallyuseful for treating infection with S. aureus, the method comprisingcontacting the substance with the nucleic acid fragment of claim of theinvention and subsequently establishing whether the substance has eitherthe ability to

-   1) bind specifically to the nucleic acid fragment, or-   2) bind specifically to a nucleic acid that hybridizes specifically    with the nucleic acid fragment.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

The term “polypeptide” is in the present context intended to mean bothshort peptides of from 2 to 10 amino acid residues, oligopeptides offrom 11 to 100 amino acid residues, and polypeptides of more than 100amino acid residues. Further-more, the term is also intended to includeproteins, i.e. functional biomolecules comprising at least onepolypeptide; when comprising at least two polypeptides, these may formcomplexes, be covalently linked, or may be non-covalently linked. Thepolypeptide (s) in a protein can be glycosylated and/or lipidated and/orcomprise prosthetic groups.

The term “subsequence” means any consecutive stretch of at least 3 aminoacids or, when relevant, of at least 3 nucleotides, derived directlyfrom a naturally occurring amino acid sequence or nucleic acid sequence,respectively

The term “amino acid sequence” is the order in which amino acidresidues, connected by peptide bonds, lie in the chain in peptides andproteins.

The term “adjuvant” has its usual meaning in the art of vaccinetechnology, i.e. a substance or a composition of matter which is 1) notin itself capable of mounting a specific immune response against theimmunogen of the vaccine, but which is 2) nevertheless capable ofenhancing the immune response against the immunogen. Or, in other words,vaccination with the adjuvant alone does not provide an immune responseagainst the immunogen, vaccination with the immunogen may or may notgive rise to an immune response against the immunogen, but the combinedvaccination with immunogen and adjuvant induces an immune responseagainst the immunogen which is stronger than that induced by theimmunogen alone.

“Sequence identity” is in the context of the present inventiondetermined by comparing 2 optimally aligned sequences of equal length(e.g. DNA, RNA or amino acid) according to the following formula:(N_(ref)−N_(dif))·100/N_(ref) wherein N_(ref) is the number of residuesin one of the 2 sequences and N_(dif) is the number of residues whichare non-identical in the two sequences when they are aligned over theirentire lengths and in the same direction. So, two sequences5′-ATTCGGAACC-3′ and 5′-ATACGGGACC-3′ will provide the sequence identity80% (N_(ref)=10 and N_(dif)=2).

An “assembly of amino acids” means two or more amino acids boundtogether by physical or chemical means.

The “3D conformation” is the 3 dimensional structure of a biomoleculesuch as a protein. In monomeric polypeptides/proteins, the 3Dconformation is also termed “the tertiary structure” and denotes therelative locations in 3 dimensional space of the amino acid residuesforming the polypeptide.

“An immunogenic carrier” is a molecule or moiety to which an immunogenor a hapten can be coupled in order to enhance or enable the elicitationof an immune response against the immunogen/hapten. Immunogenic carriersare in classical cases relatively large molecules (such as tetanustoxoid, KLH, diphtheria toxoid etc.) which can be fused or conjugated toan immunogen/hapten, which is not sufficiently immunogenic in its ownright—typically, the immunogenic carrier is capable of eliciting astrong T-helper lymphocyte response against the combined substanceconstituted by the immunogen and the immunogenic carrier, and this inturn provides for improved responses against the immunogen byB-lymphocytes and cytotoxic lymphocytes. More recently, the largecarrier molecules have to a certain extent been substituted by so-calledpromiscuous T-helper epitopes, i.e. shorter peptides that are recognizedby a large fraction of HLA haplotypes in a population, and which elicitT-helper lymphocyte responses.

A “T-helper lymphocyte response” is an immune response elicited on thebasis of a peptide, which is able to bind to an MHC class II molecule(e.g. an HLA class II molecule) in an antigen-presenting cell and whichstimulates T-helper lymphocytes in an animal species as a consequence ofT-cell receptor recognition of the complex between the peptide and theMHC Class II molecule present.

An “immunogen” is a substance of matter which is capable of inducing anadaptive immune response in a host, whose immune system is confrontedwith the immunogen. As such, immunogens are a subset of the larger genus“antigens”, which are substances that can be recognized specifically bythe immune system (e.g. when bound by antibodies or, alternatively, whenfragments of the are antigens bound to MHC molecules are beingrecognized by T-cell receptors) but which are not necessarily capable ofinducing immunity—an antigen is, however, always capable of elicitingimmunity, meaning that a host that has an established memory immunityagainst the antigen will mount a specific immune response against theantigen.

A “hapten” is a small molecule, which can neither induce nor elicit animmune response, but if conjugated to an immunogenic carrier, antibodiesor TCRs that recognize the hapten can be induced upon confrontation ofthe immune system with the hapten carrier conjugate.

An “adaptive immune response” is an immune response in response toconfrontation with an antigen or immunogen, where the immune response isspecific for antigenic determinants of the antigen/immunogen—examples ofadaptive immune responses are induction of antigen specific antibodyproduction or antigen specific induction/activation of T helperlymphocytes or cytotoxic lymphocytes.

A “protective, adaptive immune response” is an antigen-specific immuneresponse induced in a subject as a reaction to immunization (artificialor natural) with an antigen, where the immune response is capable ofprotecting the subject against subsequent challenges with the antigen ora pathology-related agent that includes the antigen. Typically,prophylactic vaccination aims at establishing a protective adaptiveimmune response against one or several pathogens.

“Stimulation of the immune system” means that a substance or compositionof matter exhibits a general, non-specific immunostimulatory effect. Anumber of adjuvants and putative adjuvants (such as certain cytokines)share the ability to stimulate the immune system. The result of using animmunostimulating agent is an increased “alertness” of the immune systemmeaning that simultaneous or subsequent immunization with an immunogeninduces a significantly more effective immune response compared toisolated use of the immunogen.

Hybridization under “stringent conditions” is herein defined ashybridization performed under conditions by which a probe will hybridizeto its target sequence, to a detectably greater degree than to othersequences. Stringent conditions are target-sequence-dependent and willdiffer depending on the structure of the polynucleotide. By controllingthe stringency of the hybridization and/or washing conditions, targetsequences can be identified which are 100% complementary to a probe(homologous probing). Alternatively, stringency conditions can beadjusted to allow some mismatching in sequences so that lower degrees ofsimilarity are detected (heterologous probing). Specificity is typicallythe function of post-hybridization washes, the critical factors beingthe ionic strength and temperature of the final wash solution.Generally, stringent wash temperature conditions are selected to beabout 5° C. to about 2° C. lower than the melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The melting point,or denaturation, of DNA occurs over a narrow temperature range andrepresents the disruption of the double helix into its complementarysingle strands. The process is described by the temperature of themidpoint of transition, Tm, which is also called the meltingtemperature. Formulas are available in the art for the determination ofmelting temperatures.

The term “animal” is in the present context in general intended todenote an animal species (preferably mammalian), such as Homo sapiens,Canis domesticus, etc. and not just one single animal. However, the termalso denotes a population of such an animal species, since it isimportant that the individuals immunized according to the method of theinvention substantially all will mount an immune response against theimmunogen of the present invention.

As used herein, the term “antibody” refers to a polypeptide or group ofpolypeptides composed of at least one antibody combining site. An“antibody combining site” is the three-dimensional binding space with aninternal surface shape and charge distribution complementary to thefeatures of an epitope of an antigen, which allows a binding of theantibody with the antigen. “Antibody” includes, for example, vertebrateantibodies, hybrid antibodies, chimeric antibodies, humanisedantibodies, altered antibodies, univalent antibodies, Fab proteins, andsingle domain antibodies.

“Specific binding” denotes binding between two substances which goesbeyond binding of either substance to randomly chosen substances andalso goes beyond simple association between substances that tend toaggregate because they share the same overall hydrophobicity orhydrophilicity. As such, specific binding usually involves a combinationof electrostatic and other interactions between two conformationallycomplementary areas on the two substances, meaning that the substancescan “recognize” each other in a complex mixture.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a heterologous nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated and expressed. Theterm further denotes certain biological vehicles useful for the samepurpose, e.g. viral vectors and phage—both these infectious agents arecapable of introducing a heterologous nucleic acid sequence

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, when the transcription product is anmRNA molecule, this is in turn translated into a protein, polypeptide,or peptide.

Specific Embodiments of the Invention

The Polypeptides of the Invention

In some embodiments the at least 5 contiguous amino acids referred to inoption b) in the definition of the first aspect of the inventionconstitute at least 6, such as at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, at least 27 at least 28, at least 29, at least 30, at least 31, atleast 32, at least 33, at least 34, and at least 35 contiguous aminoacid residues. The number can be higher, for all of SEQ ID NOs. 1-19 atleast 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, and at least 124 contiguous amino acidresidues. Another way to phrase this is that for each of SEQ ID NOs:1-19, the number of the contiguous amino acid residues is at least N−n,where N is the length of the sequence ID in question and n is anyinteger between 6 and N−1; that is, the at least 5 contiguous aminoacids can be at least any number between 5 and the length of thereference sequence minus one, in increments of one.

In some embodiments, the polypeptide of the invention also has asequence identity with the amino acid sequence of a) defined above of atleast 65%, such as at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, and atleast 99%. Similarly, the polypeptide of the invention in someembodiments also has a sequence identity with the amino acid sequence ofb) defined above of at least 60%, such as at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, and at least 99%.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, and 128 in any one of SEQ ID NOs: 1-19, if the length of theat least 5 amino acid residues so permit—if the length of the at least 5amino acids are higher than 5, the N-terminal first residue will not behigher numbered than N−L+1, where N is the number of amino acid residuesof the reference sequence and L is the number of amino acids defined foroption b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140 in anyone of SEQ ID NOs: 1, 2, and 4-19, if the length of the at least 5 aminoacid residues so permit—if the length of the at least 5 amino acids arehigher than 5, the N-terminal first residue will not be higher numberedthan N−L+1, where N is the number of amino acid residues of thereference sequence and L is the number of amino acids defined for optionb.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 141, 142, 143,144, 145, 146, 147, 148, 149, and 150 in any one of SEQ ID NOs: 1, 2,and 4-6, and 8-19, if the length of the at least 5 amino acid residuesso permit—if the length of the at least 5 amino acids are higher than 5,the N-terminal first residue will not be higher numbered than N−L+1,where N is the number of amino acid residues of the reference sequenceand L is the number of amino acids defined for option b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, and 180 inany one of SEQ ID NOs: 2, 4-6, and 8-19, if the length of the at least 5amino acid residues so permit—if the length of the at least 5 aminoacids are higher than 5, the N-terminal first residue will not be highernumbered than N−L+1, where N is the number of amino acid residues of thereference sequence and L is the number of amino acids defined for optionb.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 181, 182, 183,184, 185, and 186 in any one of SEQ ID NOs: 4-6, and 8-19, if the lengthof the at least 5 amino acid residues so permit—if the length of the atleast 5 amino acids are higher than 5, the N-terminal first residue willnot be higher numbered than N−L+1, where N is the number of amino acidresidues of the reference sequence and L is the number of amino acidsdefined for option b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,and 204 in any one of SEQ ID NOs: 4-6, 8-11, 13-19, if the length of theat least 5 amino acid residues so permit—if the length of the at least 5amino acids are higher than 5, the N-terminal first residue will not behigher numbered than N−L+1, where N is the number of amino acid residuesof the reference sequence and L is the number of amino acids defined foroption b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to amino acid residue 205 in any one of SEQ IDNOs: 4-6, 8-11, 13-15, and 17-19, if the length of the at least 5 aminoacid residues so permit—if the length of the at least 5 amino acids arehigher than 5, the N-terminal first residue will not be higher numberedthan N−L+1, where N is the number of amino acid residues of thereference sequence and L is the number of amino acids defined for optionb.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,and 223 in any one of SEQ ID NOs: 4-6, 8-10, 13-15, and 17-19, if thelength of the at least 5 amino acid residues so permit—if the length ofthe at least 5 amino acids are higher than 5, the N-terminal firstresidue will not be higher numbered than N−L+1, where N is the number ofamino acid residues of the reference sequence and L is the number ofamino acids defined for option b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 224, 225, 226,and 227 in any one of SEQ ID NOs: 4-6, 8-10, 13-15, 18, and 19, if thelength of the at least 5 amino acid residues so permit—if the length ofthe at least 5 amino acids are higher than 5, the N-terminal firstresidue will not be higher numbered than N−L+1, where N is the number ofamino acid residues of the reference sequence and L is the number ofamino acids defined for option b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, and 392 in any one of SEQ ID NOs:4-6, 8-10, 13-15, and 18, if the length of the at least 5 amino acidresidues so permit—if the length of the at least 5 amino acids arehigher than 5, the N-terminal first residue will not be higher numberedthan N−L+1, where N is the number of amino acid residues of thereference sequence and L is the number of amino acids defined for optionb.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 393, 394, 395,396, 397, 398, 399, and 400 in any one of SEQ ID NOs: 4-6, 8-10, 13, 15,and 18, if the length of the at least 5 amino acid residues so permit—ifthe length of the at least 5 amino acids are higher than 5, theN-terminal first residue will not be higher numbered than N−L+1, where Nis the number of amino acid residues of the reference sequence and L isthe number of amino acids defined for option b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 401, 402, 403,404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431,432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473,474, 475, 476, 477, 478, 479, 480, 481, and 482 in any one of SEQ IDNOs: SEQ ID NOs: 4-6, 8-10, 13, and 15, if the length of the at least 5amino acid residues so permit—if the length of the at least 5 aminoacids are higher than 5, the N-terminal first residue will not be highernumbered than N−L+1, where N is the number of amino acid residues of thereference sequence and L is the number of amino acids defined for optionb.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 483, 484, 485,486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499,500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513,514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527,528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541,542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555,556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569,570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583,584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597,598, 599, 600, 601, 602, 603, 604, and 605 in any one of SEQ ID NOs:4-6, 8, 10, 13, and 15, if the length of the at least 5 amino acidresidues so permit—if the length of the at least 5 amino acids arehigher than 5, the N-terminal first residue will not be higher numberedthan N−L+1, where N is the number of amino acid residues of thereference sequence and L is the number of amino acids defined for optionb.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 606, 607, 608,609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636,637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664,665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678,679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692,693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720,721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734,735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748,749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762,763, 764, and 765 in any one of SEQ ID NOs: 4, 5, 8, 10, 13, and 15, ifthe length of the at least 5 amino acid residues so permit—if the lengthof the at least 5 amino acids are higher than 5, the N-terminal firstresidue will not be higher numbered than N−L+1, where N is the number ofamino acid residues of the reference sequence and L is the number ofamino acids defined for option b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 766, 767, 768,769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782,783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796,797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810,811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824,825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838,839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852,853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866,867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880,881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894,895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908,909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922,923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936,937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950,951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964,965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978,979, 980, 981, 982, 983, 984, 985, 986, 987, 988, and 989 in any one ofSEQ ID NOs: 4, 5, 8, 10, and 13, if the length of the at least 5 aminoacid residues so permit—if the length of the at least 5 amino acids arehigher than 5, the N-terminal first residue will not be higher numberedthan N−L+1, where N is the number of amino acid residues of thereference sequence and L is the number of amino acids defined for optionb.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 990, 991, 992,993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, and1005, in any one of SEQ ID NOs: 5, 8, 10, and 13, if the length of theat least 5 amino acid residues so permit—if the length of the at least 5amino acids are higher than 5, the N-terminal first residue will not behigher numbered than N−L+1, where N is the number of amino acid residuesof the reference sequence and L is the number of amino acids defined foroption b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 1006, 1007,1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019,1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031,1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043,1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055,1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067,1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079,1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091,1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103,1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115,1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127,1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139,1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151,1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163,1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175,1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187,1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199,1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211,1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223,1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235,1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247,1248, 1249, 1250, 1251, 1252, and 1253 in any one of SEQ ID NOs: 5, 8,and 10, if the length of the at least 5 amino acid residues so permit—ifthe length of the at least 5 amino acids are higher than 5, theN-terminal first residue will not be higher numbered than N−L+1, where Nis the number of amino acid residues of the reference sequence and L isthe number of amino acids defined for option b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 1254, 1255,1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267,1268, 1269, and 1270 in SEQ ID NO: 5 or 10, if the length of the atleast 5 amino acid residues so permit—if the length of the at least 5amino acids are higher than 5, the N-terminal first residue will not behigher numbered than N−L+1, where N is the number of amino acid residuesof the reference sequence and L is the number of amino acids defined foroption b.

In the embodiments defined by option b) above, the polypeptide of theinvention is also one that has at least 5 contiguous amino acid residuesdefined for option b) above and also has its N-terminal amino acidresidue corresponding to any one of amino acid residues 1271, 1272,1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284,1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296,1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308,1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320,1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332,1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344,1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356,1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368,1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380,1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392,1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404,1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416,1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428,1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440,1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452,1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464,1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476,1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488,1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500,1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512,1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524,1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536,1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548,1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560,1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572,1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584,1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596,1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608,1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620,1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632,1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644,1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656,1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668,1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680,1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692,1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704,1705, 1706, 1707, 1708, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716,1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728,1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740,1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752,1753, 1754, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764,1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776,1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788,1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800,1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812,1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824,1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836,1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848,1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860,1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872,1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884,1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896,1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908,1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920,1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932,1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944,1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956,1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968,1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980,1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992,1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004,2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016,2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028,2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040,2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052,2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, and 2062 in SEQ IDNO: 5, if the length of the at least 5 amino acid residues so permit—ifthe length of the at least 5 amino acids are higher than 5, theN-terminal first residue will not be higher numbered than N−L+1, where Nis the number of amino acid residues of the reference sequence and L isthe number of amino acids defined for option b.

The polypeptide of the invention is in certain embodiments also fused orconjugated to an immunogenic carrier molecule; or, phrased otherwise,the polypeptide of the invention also includes such an immunogeniccarrier molecule in addition to the material derived from SEQ ID NOs.1-19. The immunogenic carrier molecule is a typically polypeptide thatinduces T-helper lymphocyte responses in a majority of humans, such asimmunogenic carrier proteins selected from the group consisting ofkeyhole limpet hemocyanino or a fragment thereof, tetanus toxoid or afragment thereof, diphtheria toxoid or a fragment thereof. Othersuitable carrier molecules are discussed infra.

In preferred embodiments, the polypeptide of the invention detailedabove is capable of inducing an adaptive immune response against thepolypeptide in a mammal, in particular in a human being. Preferably, theadaptive immune response is a protective adaptive immune responseagainst infection with S. aureus, in particular multi-resistant S.aureus. The polypeptide may in these cases induce a humeral and/or acellular immune response.

Epitopes

SEQ ID NOs: 1-19 include antigenic determinants (epitopes) that are assuch recognized by antibodies and/or when bound to MHC molecules byT-cell receptors. For the purposes of the present invention, B-cellepitopes (i.e. antibody binding epitopes) are of particular relevance.

It is relatively uncomplicated to identify linear B-cell epitopes—onevery simple approach entails that antibodies raised against S. aureus orS. aureus derived proteins disclosed herein are tested for binding tooverlapping oligomeric peptides derived from any one of SEQ ID NO: 1-19.Thereby, the regions of the S. aureus polypeptide which are responsiblefor or contribute to binding to the antibodies can be identified.

Alternatively, or additionally, one can produce mutated versions of thepolypeptides of the invention, e.g. version where each singlenon-alanine residue in SEQ ID NOs.: 1-19 are point mutated toalanine—this method also assists in identifying complex assembled B-cellepitopes; this is the case when binding of the same antibody is modifiedby exchanging amino acids in different areas of the full-lengthpolypeptide.

Also, in silico methods for B-cell epitope prediction can be employed:useful state-of-the-art systems for β-turn prediction is provided inPetersen B et al. (November 2010), Plos One 5(11): e15079; prediction oflinear B-cell epitopes, cf: Larsen J E P et al. (April 2006), ImmunomeResearch, 2:2; prediction of solvent exposed amino acids: Petersen B etal (July 2009), BMC Structural Biology, 9:51.

The Nucleic Acid Fragments of the Invention

The nucleic acid fragment of the invention referred to above ispreferably is a DNA fragment (such as SEQ ID NOs: 20-38) or an RNAfragment (such as SEQ ID NOs 29-58).

The nucleic acid fragment of the invention typically consists of atleast 11, such as at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17 at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, at least43, at least 44, at least 45, at least 46, at least 47, at least 48, atleast 49, at least 50, at least 51, at least 52, at least 53, at least54, at least 55, at least 56, at least 57, at least 58, at least 59, atleast 60, at least 61, at least 62, at least 63, at least 64, at least65, at least 66, at least 67, at least 68, at least 69, at least 70, atleast 71, at least 72, at least 73, at least 74, at least 75, at least76, at least 77, at least 78, at least 79, at least 80, at least 81, atleast 82, at least 83, at least 84, at least 85, at least 86, at least87, at least 88, at least 89, at least 90, at least 91, at least 92, atleast 93, at least 94, at least 95, at least 96, at least 97, at least98, at least 99, at least 100, at least 101, at least 102, at least 103,at least 104, at least 105, at least 106, at least 107, at least 108, atleast 109, at least 110, at least 111, at least 112, at least 113, atleast 114, at least 115, at least 116, at least 117, at least 118, atleast 119, at least 120, at least 121, at least 122, at least 123, atleast 124, at least 125, at least 126, at least 127, at least 128, atleast 129, at least 130, at least 131, at least 132, at least 133, atleast 134, at least 135, at least 136, at least 137, at least 138, atleast 139, at least 140, at least 141, at least 142, at least 143, atleast 144, at least 145, at least 146, at least 147, at least 148, atleast 149, at least 150, at least 151, at least 152, at least 153, atleast 154, at least 155, at least 156, at least 157, at least 158, atleast 159, at least 160, at least 161, at least 162, at least 163, atleast 164, at least 165, at least 166, at least 167, at least 168, atleast 169, at least 170, at least 171, at least 172, at least 173, atleast 174, at least 175, at least 176, at least 177, at least 178, atleast 179, at least 180, at least 181, at least 182, at least 183, atleast 184, at least 185, at least 186, at least 187, at least 188, atleast 189, at least 190, at least 191, at least 192, at least 193, atleast 194, at least 195, at least 196, at least 197, at least 198, atleast 199, at least 200, at least 201, at least 202, at least 203, atleast 204, at least 205, at least 206, at least 207, at least 208, atleast 209, at least 210, at least 211, at least 212, at least 213, atleast 214, at least 215, at least 216, at least 217, at least 218, atleast 219, at least 220, at least 221, at least 222, at least 223, atleast 224, at least 225, at least 226, at least 227, at least 228, atleast 229, at least 230, at least 231, at least 232, at least 233, atleast 234, at least 235, at least 236, at least 237, at least 238, atleast 239, at least 240, at least 241, at least 242, at least 243, atleast 244, at least 245, at least 246, at least 247, at least 248, atleast 249, at least 250, at least 251, at least 252, at least 253, atleast 254, at least 255, at least 256, at least 257, at least 258, atleast 259, at least 260, at least 261, at least 262, at least 263, atleast 264, at least 265, at least 266, at least 267, at least 268, atleast 269, at least 270, at least 271, at least 272, at least 273, atleast 274, at least 275, at least 276, at least 277, at least 278, atleast 279, at least 280, at least 281, at least 282, at least 283, atleast 284, at least 285, at least 286, at least 287, at least 288, atleast 289, at least 290, at least 291, at least 292, at least 293, atleast 294, at least 295, at least 296, at least 297, at least 298, atleast 299, at least 300, at least 301, at least 302, at least 303, atleast 304, at least 305, at least 306, at least 307, at least 308, atleast 309, at least 310, at least 311, at least 312, at least 313, atleast 314, at least 315, at least 316, at least 317, at least 318, atleast 319, at least 320, at least 321, at least 322, at least 323, atleast 324, at least 325, at least 326, at least 327, at least 328, atleast 329, at least 330, at least 331, at least 332, at least 333, atleast 334, at least 335, at least 336, at least 337, at least 338, atleast 339, at least 340, at least 341, at least 342, at least 343, atleast 344, at least 345, at least 346, at least 347, at least 348, atleast 349, at least 350, at least 351, at least 352, at least 353, atleast 354, at least 355, at least 356, at least 357, at least 358, atleast 359, at least 360, at least 361, at least 362, at least 363, atleast 364, at least 365, at least 366, at least 367, at least 368, atleast 369, at least 370, at least 371, at least 372, at least 373, atleast 374, at least 375, at least 376, at least 377, at least 378, atleast 379, at least 380, at least 381, at least 382, at least 383, atleast 384, at least 385, at least 386, at least 387 consecutivenucleotides in any one of SEQ ID NOs: 20-57. Longer fragments arecontemplated, i.e. fragments having at least 400, at least 500, at least600, at least 700, at least 800, at least 900, at least 1000, at least1500, at least 2000, and at least 2500 nucleotides from those of SEQ IDNOs: 20-57 that encompass fragments of such lengths.

The nucleic acid fragment of the invention discussed above typically hasa sequence identity with the nucleotide sequence defined for i) or ii)above, which is at least 65%, such as at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, and at least 99%.

The nucleic acid fragment of the invention discussed above may also havea sequence identity with the nucleotide sequence defined for iii) above,which is at least 65%, such as at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, andat least 99%.

The Vectors of the Invention

Vectors of the invention fall into several categories discussed infra.One preferred vector of the invention comprises in operable linkage andin the 5′-3′ direction, an expression control region comprising anenhancer/promoter for driving expression of the nucleic acid fragmentdefined for option i) above, optionally a signal peptide codingsequence, a nucleotide sequence defined for option i), and optionally aterminator. Hence, such a vector constitutes an expression vector usefulfor effecting production in cells of the polypeptide of the invention.Since the polypeptides of the invention are bacterial of origin,recombinant production is conveniently effected in bacterial host cells,so here it is preferred that the expression control region drivesexpression in prokaryotic cell such as a bacterium, e.g. in E coli.However, if the vector is to drive expression in mammalian cell (aswould be the case for a DNA vaccine vector), the expression controlregion should be adapted to this particular use.

At any rate, certain vectors of the invention are capable of autonomousreplication.

Also, the vector of the invention may be one that is capable of beingintegrated into the genome of a host cell—this is particularly useful ifthe vector is use in the production of stably transformed cells, wherethe progeny will also include the genetic information introduced via thevector. Alternatively, vectors incapable of being integrated into thegenome of a mammalian host cell are useful in e.g. DNA vaccination.

Typically, the vector of the invention is selected from the groupconsisting of a virus, such as an attenuated virus (which may in itselfbe useful as a vaccine agent), a bacteriophage, a plasmid, aminichromosome, and a cosmid.

A more detailed discussion of vectors of the invention is provided inthe following:

Polypeptides of the invention may be encoded by a nucleic acid moleculecomprised in a vector. A nucleic acid sequence can be “heterologous,”which means that it is in a context foreign to the cell in which thevector is being introduced, which includes a sequence homologous to asequence in the cell but in a position within the host cell where it isordinarily not found. Vectors include naked DNAs, RNAs, plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (for example Sambrook et al, 2001; Ausubel et al, 1996, bothincorporated herein by reference). In addition to encoding thepolypeptides of this invention, a vector of the present invention mayencode polypeptide sequences such as a tag or immunogenicity enhancingpeptide (e.g. an immunogenic carrier or a fusion partner that stimulatesthe immune system, such as a cytokine or active fragment thereof).Useful vectors encoding such fusion proteins include pIN vectors (Inouyeet al, 1985), vectors encoding a stretch of histidines, and pGEXvectors, for use in generating glutathione S-transferase (GST) solublefusion proteins for later purification and separation or cleavage.

Vectors of the invention may be used in a host cell to produce apolypeptide of the invention that may subsequently be purified foradministration to a subject or the vector may be purified for directadministration to a subject for expression of the protein in the subject(as is the case when administering a nucleic acid vaccine).

Expression vectors can contain a variety of “control sequences,” whichrefer to nucleic acid sequences necessary for the transcription andpossibly translation of an operably linked coding sequence in aparticular host organism. In addition to control sequences that governtranscription and translation, vectors and expression vectors maycontain nucleic acid sequences that serve other functions as well andare described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence. The promoter is typically a regionof a nucleic acid sequence at which initiation and rate of transcriptionare controlled. It may contain genetic elements at which regulatoryproteins and molecules may bind such as RNA polymerase and othertranscription factors. The phrases “operatively positioned,”“operatively linked,” “under control,” and “under transcriptionalcontrol” mean that a promoter is in a correct functional location and/ororientation in relation to a nucleic acid sequence to controltranscriptional initiation and expression of that sequence. A promotermay or may not be used in conjunction with an “enhancer,” which refersto a cis-acting regulatory sequence involved in the transcriptionalactivation of a nucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment or exon. Such a promoter can be referredto as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural state. Such promoters or enhancers may include promoters orenhancers of other genes, and promoters or enhancers isolated from anyother prokaryotic, viral, or eukaryotic cell, and promoters or enhancersnot “naturally occurring,” i.e., containing different elements ofdifferent transcriptional regulatory regions, and/or mutations thatalter expression. In addition to producing nucleic acid sequences ofpromoters and enhancers synthetically, sequences may be produced usingrecombinant cloning and/or nucleic acid amplification technology,including PCR™, in connection with the compositions disclosed herein(see U.S. Pat. Nos. 4,683,202; 5,928,906, each incorporated herein byreference).

Naturally, it may be important to employ a promoter and/or enhancer thateffectively direct(s) the expression of the DNA segment in the cell typeor organism chosen for expression. Those of skill in the art ofmolecular biology generally know the use of promoters, enhancers, andcell type combinations for protein expression (see Sambrook et al, 2001,incorporated herein by reference). The promoters employed may beconstitutive, tissue-specific, or inducible and in certain embodimentsmay direct high level expression of the introduced DNA segment underspecified conditions, such as large-scale production of recombinantproteins or peptides.

Examples of inducible elements, which are regions of a nucleic acidsequence that can be activated in response to a specific stimulus,include but are not limited to Immunoglobulin Heavy Chain (Banerji etal, 1983; Gilles et al, 1983; Grosschedl et al, 1985; Atchinson et al,1986, 1987; toiler et al, 1987; Weinberger et al, 1984; Kiledjian et al,1988; Porton et al; 1990), Immunoglobulin Light Chain (Queen et al,1983; Picard et al, 1984), T Cell Receptor (Luria et al, 1987; Winoto etal, 1989; Redondo et al; 1990), HLA DQα and/or DQβ (Sullivan et al,1987), β-Interferon (Goodbourn et al, 1986; Fujita et al, 1987;Goodbourn et al, 1988), Interleukin-2 (Greene et al, 1989),Interleukin-2 Receptor (Greene et al, 1989; Lin et al, 1990), MHC ClassII 5 (Koch et al, 1989), MHC Class II HLA-DRα (Sherman et al, 1989),β-Actin (Kawamoto et al, 1988; Ng et al; 1989), Muscle Creatine Kinase(MCK) (Jaynes et al, 1988; Horlick et al, 1989; Johnson et al, 1989),Prealbumin (Transthyretin) (Costa et al, 1988), Elastase I (Omitz et al,1987), Metallothionein (MTII) (Karin et al, 1987; Culotta et al, 1989),Collagenase (Pinkert et al, 1987; Angel et al, 1987), Albumin (Pinkertet al, 1987; Tranche et al, 1989, 1990), α-Fetoprotein (Godbout et al,1988; Campere et al, 1989), γ-Globin (Bodine et al, 1987; Perez-Stableet al, 1990), β-Globin (Trudel et al, 1987), c-fos (Cohen et al, 1987),c-HA-ras (Triesman, 1986; Deschamps et al, 1985), Insulin (Edlund et al,1985), Neural Cell Adhesion Molecule (NCAM) (Hirsh et al, 1990),αI-Antitrypain (Larimer et al, 1990), H2B (TH2B) Histone (Hwang et al,1990), Mouse and/or Type I Collagen (Ripe et al, 1989),Glucose-Regulated Proteins (GRP94 and GRP78) (Chang et al, 1989), RatGrowth Hormone (Larsen et al, 1986), Human Serum Amyloid A (SAA)(Edbrooke et al, 1989), Troponin I (TN I) (Yutzey et al, 1989),Platelet-Derived Growth Factor (PDGF) (Pech et al, 1989), DuchenneMuscular Dystrophy (Klamut et al, 1990), SV40 (Banerji et al, 1981;Moreau et al, 1981; Sleigh et al, 1985; Firak et al, 1986; Herr et al,1986; Imbra et al, 1986; Kadesch et al, 1986; Wang et al, 1986; Ondek etal, 1987; Kuhl et al, 1987; Schaffner et al, 1988), Polyoma(Swartzendruber et al, 1975; Vasseur et al, 1980; Katinka et al, 1980,1981; Tyndell et al, 1981; Dandolo et al, 1983; de Villiers et al, 1984;Hen et al, 1986; Satake et al, 1988; Campbell et al, 1988), Retroviruses(Kriegler et al, 1982, 1983; Levinson et al, 1982; Kriegler et al, 1983,1984a, b, 1988; Bosze et al, 1986; Miksicek et al, 1986; Celander et al,1987; Thiesen et al, 1988; Celander et al, 1988; Choi et al, 1988;Reisman et al, 1989), Papilloma Virus (Campo et al, 1983; Lusky et al,1983; Spandidos and Wilkie, 1983; Spalholz et al, 1985; Lusky et al,1986; Cripe et al, 1987; Gloss et al, 1987; Hirochika et al, 1987;Stephens et al, 1987), Hepatitis B Virus (Bulla et al, 1986; Jameel etal, 1986; Shaul et al, 1987; Spandau et al, 1988; Vannice et al, 1988),Human Immunodeficiency Virus (Muesing et al, 1987; Hauber et al, 1988;Jakobovits et al, 1988; Feng et al, 1988; Takebe et al, 1988; Rosen etal, 1988; Berkhout et al, 1989; Laspia et al, 1989; Sharp et al, 1989;Braddock et al, 1989), Cytomegalovirus (CMV) IE (Weber et al, 1984;Boshart et al, 1985; Foecking et al, 1986), Gibbon Ape Leukemia Virus(Holbrook et al, 1987; Quinn et al, 1989).

Inducible Elements include, but are not limited to MT II—Phorbol Ester(TFA)/Heavy metals (Palmiter et al, 1982; Haslinger et al, 1985; Searleet al, 1985; Stuart et al, 1985; Imagawa et al, 1987, Karin et al, 1987;Angel et al, 1987b; McNeall et al, 1989); MMTV (mouse mammary tumorvirus)—Glucocorticoids (Huang et al, 1981; Lee et al, 1981; Majors etal, 1983; Chandler et al, 1983; Lee et al, 1984; Ponta et al, 1985;Sakai et al, 1988); β-Interferon—poly(rI)x/poly(rc) (Tavernier et al,1983); Adenovirus 5 E2—EIA (Imperiale et al, 1984); Collagenase—PhorbolEster (TPA) (Angel et al, 1987a); Stromelysin—Phorbol Ester (TPA) (Angelet al, 1987b); SV40—Phorbol Ester (TPA) (Angel et al, 1987b); Murine MXGene—Interferon, Newcastle Disease Virus (Hug et al, 1988); GRP78Gene—A23187 (Resendez et al, 1988); α-2-Macroglobulin—IL-6 (Kunz et al,1989); Vimentin—Serum (Rittling et al, 1989); MHC Class I GeneH-2κb—Interferon (Blanar et al, 1989); HSP70—E1A/SV40 Large T Antigen(Taylor et al, 1989, 1990a, 1990b); Proliferin—Phorbol Ester/TPA(Mordacq et al, 1989); Tumor Necrosis Factor—PMA (Hensel et al, 1989);and Thyroid Stimulating Hormonea Gene—Thyroid Hormone (Chatterjee et al,1989).

Also contemplated as useful in the present invention are the dectin-1and dectin-2 promoters. Additionally any promoter/enhancer combination(as per the Eukaryotic Promoter Data Base EPDB) could also be used todrive expression of structural genes encoding oligosaccharide processingenzymes, protein folding accessory proteins, selectable marker proteinsor a heterologous protein of interest.

The particular promoter that is employed to control the expression ofpeptide or protein encoding polynucleotide of the invention is notbelieved to be critical, so long as it is capable of expressing thepolynucleotide in a targeted cell, preferably a bacterial cell. Where ahuman cell is targeted, it is preferable to position the polynucleotidecoding region adjacent to and under the control of a promoter that iscapable of being expressed in a human cell. Generally speaking, such apromoter might include either a bacterial, human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, and the Rous sarcoma virus longterminal repeat can be used to obtain high level expression of a relatedpolynucleotide to this invention. The use of other viral or mammaliancellular or bacterial phage promoters, which are well known in the art,to achieve expression of polynucleotides is contemplated as well.

In embodiments in which a vector is administered to a subject forexpression of the protein, it is contemplated that a desirable promoterfor use with the vector is one that is not down-regulated by cytokinesor one that is strong enough that even. if down-regulated, it producesan effective amount of the protein/polypeptide of the current inventionin a subject to elicit an immune response. Non-limiting examples ofthese are CMV IE and RSV LTR. In other embodiments, a promoter that isup-regulated in the presence of cytokines is employed. The MHC Ipromoter increases expression in the presence of IFN-γ.

Tissue specific promoters can be used, particularly if expression is incells in which expression of an antigen is desirable, such as dendriticcells or macrophages. The mammalian MHC I and MHC II promoters areexamples of such tissue-specific promoters. 2. Initiation Signals andInternal Ribosome Binding Sites (IRES)

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic and may be operable in bacteria or mammaliancells. The efficiency of expression may be enhanced by the inclusion ofappropriate transcription enhancer elements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well as an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

2. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. (See Carbonelli et al, 1999, Levenson et al, 1998,and Cocea, 1997, incorporated herein by reference.) Frequently, a vectoris linearized or fragmented using a restriction enzyme that cuts withinthe MCS to enable exogenous sequences to be ligated to the vector.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

3. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. If relevant in the contextof vectors of the present invention, vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al, 1997, incorporated herein by reference.)

4. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (poly A)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, the bovinegrowth hormone terminator or viral termination sequences, such as theSV40 terminator. In certain embodiments, the termination signal may be alack of transcribable or translatable sequence, such as due to asequence truncation.

5. Polyadenylation Signals

In expression, particularly eukaryotic expression (as is relevant innucleic acid vaccination), one will typically include a polyadenylationsignal to effect proper polyadenylation of the transcript. The nature ofthe polyadenylation signal is not believed to be crucial to thesuccessful practice of the invention, and/or any such sequence may beemployed. Preferred embodiments include the SV40 polyadenylation signaland/or the bovine growth hormone polyadenylation signal, convenientand/or known to function well in various target cells. Polyadenylationmay increase the stability of the transcript or may facilitatecytoplasmic transport.

6. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “on”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

7. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby encoding a screenable or selectable marker in the expression vector.When transcribed and translated, a marker confers an identifiable changeto the cell permitting easy identification of cells containing theexpression vector. Generally, a selectable marker is one that confers aproperty that allows for selection. A positive selectable marker is onein which the presence of the marker allows for its selection, while anegative selectable marker is one in which its presence prevents itsselection. An example of a positive selectable marker is a drugresistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, markers that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin orhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP for colorimetricanalysis. Alternatively, screenable enzymes such as herpes simplex virusthymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may beutilized. One of skill in the art would also know how to employimmunologic markers that can be used in conjunction with FACS analysis.The marker used is not believed to be important, so long as it iscapable of being expressed simultaneously with the nucleic acid encodinga protein of the invention. Further examples of selectable andscreenable markers are well known to one of skill in the art.

The Transformed Cells of the Invention

Transformed cells of the invention are useful as organisms for producingthe polypeptide of the invention, but also as simple “containers” ofnucleic acids and vectors of the invention.

Certain transformed cells of the invention are capable of replicatingthe nucleic acid fragment defined for option i) of the second aspect ofthe invention. Preferred transformed cells of the invention are capableof expressing the nucleic acid fragment defined for option i).

For recombinant production it is convenient, but not a prerequisite thatthe transformed cell according is prokaryotic, such as a bacterium, butgenerally both prokaryotic cells and eukaryotic cells may be used.

Suitable prokaryotic cells are bacterial cells selected from the groupconsisting of Escherichia (such as E. coli.), Bacillus [e.g. Bacillussubtilis], Salmonella, and Mycobacterium [preferably non-pathogenic,e.g. M. bovis BCG].

Eukaryotic cells can be in the form of yeasts (such as Saccharomycescerevisiae) and protozoans. Alternatively, the transformed eukaryoticcells are derived from a multicellular organism such as a fungus, aninsect cell, a plant cell, or a mammalian cell.

For production purposes, it is advantageous that the transformed cell ofthe invention is stably transformed by having the nucleic acid definedabove for option i) stably integrated into its genome, and in certainembodiments it is also preferred that the transformed cell secretes orcarries on its surface the polypeptide of the invention, since thisfacilitates recovery of the polypeptides produced. A particular versionof this embodiment is one where the transformed cell is a bacterium andsecretion of the polypeptide of the invention is into the periplasmicspace.

As noted above, stably transformed cells are preferred—these inter a/ia(i.a.) allows that cell lines comprised of transformed cells as definedherein may be established—such cell lines are particularly preferredaspects of the invention.

Further details on cells and cell lines are presented in the following:

Suitable cells for recombinant nucleic acid expression of the nucleicacid fragments of the present invention are prokaryotes and eukaryotes.Examples of prokaryotic cells include E. coli; members of theStaphylococcus genus, such as S. epidermidis; members of theLactobacillus genus, such as L. plantarum; members of the Lactococcusgenus, such as L. lactis; members of the Bacillus genus, such as B.subtilis; members of the Corynebacterium genus such as C. glutamicum;and members of the Pseudomonas genus such as Ps. fluorescens. Examplesof eukaryotic cells include mammalian cells; insect cells; yeast cellssuch as members of the Saccharomyces genus (e.g. S. cerevisiae), membersof the Pichia genus (e.g. P. pastoris), members of the Hansenula genus(e.g. H. polymorpha), members of the Kluyveromyces genus (e.g. K. lactisor K. fragilis) and members of the Schizosaccharomyces genus (e.g. S.pombe).

Techniques for recombinant gene production, introduction into a cell,and recombinant gene expression are well known in the art. Examples ofsuch techniques are provided in references such as Ausubel, CurrentProtocols in Molecular Biology, John Wiley, 1987-2002, and Sambrook etal., Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold SpringHarbor Laboratory Press, 1989.

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors or viruses. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid, such as a recombinant protein-encoding sequence,is transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, includingbacteria, yeast cells, insect cells, and mammalian cells for replicationof the vector or expression of part or all of the nucleic acidsequence(s). Numerous cell lines and cultures are available for use as ahost cell, and they can be obtained through the American Type CultureCollection (ATCC), which is an organization that serves as an archivefor living cultures and genetic materials (www.atcc.org) or from otherdepository institutions such as Deutsche Sammlung vor Micrroorganismenand Zellkulturen (DSM). An appropriate host can be determined by one ofskill in the art based on the vector backbone and the desired result. Aplasmid or cosmid, for example, can be introduced into a prokaryote hostcell for replication of many vectors or expression of encoded proteins.Bacterial cells used as host cells for vector replication and/orexpression include Staphylococcus strains, DH5α, JMI 09, and KC8, aswell as a number of commercially available bacterial hosts such as SURE®Competent Cells and SOLOP ACK™ Gold Cells (STRATAGENE®, La Jolla,Calif.). Alternatively, bacterial cells such as E. coli LE392 could beused as host cells for phage viruses. Appropriate yeast cells includeSaccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ Baculovirus expression system fromCLONTECH®.

In addition to the disclosed expression systems of the invention, otherexamples of expression systems include STRATAGENE®'s COMPLETE CONTROL™Inducible Mammalian Expression System, which involves a syntheticecdysone-inducible receptor, or its pET Expression System, an E. coliexpression system. Another example of an inducible expression system isavailable from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al, 2001). In certain embodiments, analysis is performed onwhole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to sequences of genes identified herein are contacted withthe template nucleic acid under conditions that permit selectivehybridization. Depending upon the desired application, high stringencyhybridization conditions may be selected that will only allowhybridization to sequences that are completely complementary to theprimers. In other embodiments, hybridization may occur under reducedstringency to allow for amplification of nucleic acids containing one ormore mismatches with the primer sequences. Once hybridized, thetemplate-primer complex is contacted with one or more enzymes thatfacilitate template-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced.

The amplification product may be detected or quantified. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electricaland/or thermal impulse signals (Bellus, 1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety.

Methods of Gene Transfer

Suitable methods for nucleic acid delivery to effect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnonviral vectors) can be introduced into a cell, a tissue or anorganism, as described herein or as would be known to one of ordinaryskill in the art. Such methods include, but are not limited to, directdelivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harland and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al, 1987); by liposome mediated transfection (Nicolau and Sene, 1982;Fraley et al, 1979; Nicolau et al, 1987; Wong et al, 1980; Kaneda et al,1989; Kato et al, 1991); by microprojectile bombardment (PCT ApplicationNos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,7835,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporatedherein by reference); by agitation with silicon carbide fibers (Kaeppleret al, 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporatedherein by reference); by Agrobacterium mediated transformation (U.S.Pat. Nos. 5,591,616 and 5,563,055, each incorporated herein byreference); or by PEG mediated transformation of protoplasts (Omirullehet al, 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporatedherein by reference); by desiccation/inhibition mediated DNA uptake(Potrykus et al, 1985). Through the application of techniques such asthese, organelle(s), cell(s), tissue(s) or organism(s) may be stably ortransiently transformed.

The Antibodies of the Invention—and their Production/Isolation

Antibodies directed against the proteins of the invention are useful foraffinity chromatography, immunoassays, and fordistinguishing/identifying staphylococcus proteins as well as forpassive immunisation and therapy.

Antibodies to the proteins of the invention, both polyclonal andmonoclonal, may be prepared by conventional methods. In general, theprotein is first used to immunize a suitable animal, preferably a mouse,rat, rabbit or goat. Rabbits and goats are preferred for the preparationof polyclonal sera due to the volume of serum obtainable, and theavailability of labeled anti-rabbit and anti-goat antibodies.Immunization is generally performed by mixing or emulsifying the proteinin saline, preferably in an adjuvant such as Freund's complete adjuvant,and injecting the mixture or emulsion parenterally (generallysubcutaneously or intramuscularly). A dose of 50-200 μg/injection istypically sufficient. Immunization is generally boosted 2-6 weeks laterwith one or more injections of the protein in saline, preferably usingFreund's incomplete adjuvant. One may alternatively generate antibodiesby in vitro immunization using methods known in the art, which for thepurposes of this invention is considered equivalent to in vivoimmunization. Polyclonal antiserum is obtained by bleeding the immunizedanimal into a glass or plastic container, incubating the blood at 25 Cfor one hour, followed by incubating at 4 C for 2-18 hours. The serum isrecovered by centrifugation (e.g. 1,000 g for 10 minutes). About 20-50ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the standard method of Kohler &Milstein [Nature (1975) 256: 495-96], or a modification thereof.Typically, a mouse or rat is immunized as described above. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) is removed and dissociated intosingle cells. If desired, the spleen cells may be screened (afterremoval of nonspecifically adherent cells) by applying a cell suspensionto a plate or well coated with the protein antigen. B-cells expressingmembrane-bound immunoglobulin specific for the antigen bind to theplate, and are not rinsed away with the rest of the suspension.Resulting B-cells, or all dissociated spleen cells, are then induced tofuse with myeloma cells to form hybridomas, and are cultured in aselective medium (e.g. hypexanthine, aminopterin, thymidine medium,“HAT”). The resulting hybridomas are plated by limiting dilution, andare assayed for production of antibodies, which bind specifically to theimmunizing antigen (and which do not bind to unrelated antigens). Theselected MAb-secreting hybridomas are then cultured either in vitro(e.g. in tissue culture bottles or hollow fiber reactors), or in vivo(as ascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may belabeled using conventional techniques. Suitable labels includefluorophores, chromophores, radioactive atoms (particularly 32p and125I), electron-dense reagents, enzymes, and ligands having specificbinding partners. Enzymes are typically detected by their activity. Forexample, horseradish peroxidase is usually detected by its ability toconvert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment,quantifiable with a spectrophotometer. “Specific binding partner” refersto a protein capable of binding a ligand molecule with high specificity,as for example in the case of an antigen and a monoclonal antibodyspecific therefor. Other specific binding partners include biotin andavidin or streptavidin, IgG and protein A, and the numerousreceptor-ligand couples known in the art. It should be understood thatthe above description is not meant to categorize the various labels intodistinct classes, as the same label may serve in several differentmodes. For example, 1151 may serve as a radioactive label or as anelectron-dense reagent. HRP may serve as enzyme or as antigen for a MAb.Further, one may combine various labels for desired effect. For example,MAbs and avidin also require labels in the practice of this invention:thus, one might label a MAb with biotin, and detect its presence withavidin labeled with, 125I, or with an anti-biotin MAb labeled with HRP.Other permutations and possibilities will be readily apparent to thoseof ordinary skill in the art, and are considered as equivalents withinthe scope of the instant invention.

According to the invention, the isolated monoclonal antibody or antibodyanalogue is preferably a monoclonal antibody selected from amulti-domain antibody such as a murine antibody, a chimeric antibodysuch as a humanized antibody, a fully human antibody, and single-domainantibody of a llama or a camel, or which is an antibody analogueselected from a fragment of an antibody such as an Fab or an F(ab′)₂, anscFV; cf. also the definition of the term “antibody” presented above.

Compositions of the Invention; Vaccines

Pharmaceutical compositions, in particular vaccines, according to theinvention may either be prophylactic (i.e. to prevent infection) ortherapeutic (i.e. to treat disease after infection).

Such vaccines comprise immunising antigen(s), immunogen(s),polypeptide(s), protein(s) or nucleic acid(s), usually in combinationwith “pharmaceutically acceptable carriers”, which include any carrierthat does not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes), and inactive virus particles.

Such carriers are well known to those of ordinary skill in the art.Additionally, these carriers may function as immunostimulating agents(“adjuvants”). Furthermore, the antigen or immunogen may be conjugatedto a bacterial toxoid, such as a toxoid from diphtheria, tetanus,cholera, H. pylori, etc. pathogen, cf. the description of immunogeniccarriers supra.

The pharmaceutical compositions of the invention thus typically containan immunological adjuvant, which is commonly an aluminium based adjuvantor one of the other adjuvants described in the following:

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: (1) aluminum salts (alum), such as aluminumhydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-wateremulsion formulations (with or without other specific immunostimulatingagents such as muramyl peptides (see below) or bacterial cell wallcomponents), such as for example (a) MF59 (WO 90/14837; Chapter 10 inVaccine design: the subunit and adjuvant approach, eds. Powell & Newman,Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span85 (optionally containing various amounts of MTP-PE (see below),although not required) formulated into submicron particles using amicrofluidizer such as Model 110Y microfluidizer (Microfluidics, Newton,Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%pluronic-blocked polymer L121, and thr-MDP (see below) eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi adjuvant system (RAS), (RibiImmunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, andone or more bacterial cell wall components from the group consisting ofmonophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants suchas Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may be used orparticles generated therefrom such as ISCOMs (immunostimulatingcomplexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA); (5) cytokines, such as interleukins (e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. gammainterferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; and (6) other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition. Alum and MF59™ adjuvants are preferred.

As mentioned above, muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2″-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

The immunogenic compositions (e.g. the immunising antigen or immunogenor polypeptide or protein or nucleic acid, pharmaceutically acceptablecarrier, and adjuvant) typically will contain diluents, such as water,saline, glycerol, ethanol, etc. Additionally, auxiliary substances, suchas wetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect, as discussed above underpharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of the antigenic or immunogenic polypeptides, as wellas any other of the above-mentioned components, as needed. By“immunollogically effective amount”, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, the taxonomic group of individual to be treated (e.g. nonhumanprimate, primate, etc.), the capacity of the individual's immune systemto synthesize antibodies or generally mount an immune response, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.However, for the purposes of protein vaccination, the amountadministered per immunization is typically in the range between 0.5 μgand 500 mg (however, often not higher than 5,000 μg), and very often inthe range between 10 and 200 μg.

The immunogenic compositions are conventionally administeredparenterally, e.g. by injection, either subcutaneously, intramuscularly,or transdermally/transcutaneously (e.g. W098/20734). Additionalformulations suitable for other modes of administration include oral andpulmonary formulations, suppositories, and transdermal applications. Inthe case of nucleic acid vaccination, also the intravenous orintraarterial routes may be applicable.

Dosage treatment may be a single dose schedule or a multiple doseschedule. The vaccine may be administered in conjunction with otherimmunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination (alsotermed nucleic acid vaccination or gene vaccination) may be used [e.g.Robinson & Torres (1997) Seminars in Immunol 9: 271-283; Donnelly et al.(1997) Avnu Rev Immunol 15: 617-648; later herein].

Treatment Methods of the Invention

The method of the sixth aspect of the invention generally relates toinduction of immunity and as such also entails method that relate totreatment, prophylaxis and amelioration of disease.

When immunization methods entail that a polypeptide of the invention ora composition comprising such a polypeptide is administered the animal(e.g. the human) typically receives between 0.5 and 5,000 μg of thepolypeptide of the invention per administration.

In preferred embodiments of the sixth aspect, the immunization schemeincludes that the animal (e.g. the human) receives a primingadministration and one or more booster administrations.

Preferred embodiments of the 6^(th) aspect of the invention comprisethat the administration is for the purpose of inducing protectiveimmunity against S. aureus. In this embodiment it is particularlypreferred that the protective immunity is effective in reducing the riskof attracting infection with S. aureus or is effective in treating orameliorating infection with S. aureus.

As mention herein, the preferred vaccines of the invention inducehumoral immunity, so it is preferred that the administration is for thepurpose of inducing antibodies specific for S. aureus and wherein saidantibodies or B-lymphocytes producing said antibodies are subsequentlyrecovered from the animal.

But, as also mentioned the method of the 6^(th) aspect may also beuseful in antibody production, so in other embodiments theadministration is for the purpose of inducing antibodies specific for S.aureus and wherein B-lymphocytes producing said antibodies aresubsequently recovered from the animal and used for preparation ofmonoclonal antibodies.

Pharmaceutical compositions can as mentioned above comprisepolypeptides, antibodies, or nucleic acids of the invention. Thepharmaceutical compositions will comprise a therapeutically effectiveamount thereof.

The term “therapeutically effective amount” or “prophylacticallyeffective amount” as used herein refers to an amount of a therapeuticagent to treat, ameliorate, or prevent a desired disease or condition,or to exhibit a detectable therapeutic or preventative effect. Theeffect can be detected by, for example, chemical markers or antigenlevels. Therapeutic effects also include reduction in physical symptoms,such as decreased body temperature. The precise effective amount for asubject will depend upon the subject's size and health, the nature andextent of the condition, and the therapeutics or combination oftherapeutics selected for administration. Thus, it is not useful tospecify an exact effective amount in advance. Reference is however madeto the ranges for dosages of immunologically effective amounts ofpolypeptides, cf. above.

However, the effective amount for a given situation can be determined byroutine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be fromabout 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNAconstructs in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, and other therapeutic agents. Theterm refers to any pharmaceutical carrier that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, and which may be administered without undue toxicity.Suitable carriers may be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art.

Pharmaceutically acceptable salts can be used therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. A thorough discussionof pharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions maycontain liquids such as water, saline, glycerol and ethanol.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles.

Typically, the therapeutic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. Liposomes are included within the definition of apharmaceutically acceptable carrier.

As is apparent from the claim, the invention also relates to relatedembodiments to the treatment and prophylaxis disclosed herein: theinvention also includes embodiments where

-   -   the polypeptide of the invention is for use as a pharmaceutical,        in particular for use as a pharmaceutical in the treatment,        prophylaxis or amelioration of infection with S. aureus;    -   the nucleic acid fragment of the invention or the vector of the        invention is for use as a pharmaceutical, in particular for use        as a pharmaceutical in the treatment, prophylaxis or        amelioration of infection with S. aureus;    -   the transformed cell of the invention is for use as a        pharmaceutical, in particular for use as a pharmaceutical in the        treatment, prophylaxis or amelioration of infection with S.        aureus.    -   the antibody, antibody fragment or antibody analogue of the        invention is for use as a pharmaceutical, in particular for use        as a pharmaceutical in the treatment, prophylaxis or        amelioration of infection with S. aureus.

EXAMPLE Protocol for Testing S. aureus Derived Vaccines in Mice

Expression and Purification of S. aureus Genes

-   1. Gene fragments that encode the selected S. aureus polypeptides of    the invention are prepared synthetically and are introduced into the    pQE-1 vector (Qiagen) from Genscript. The fragments are inserted by    blunt ended ligation into the PVU II site in the 5′-end, immediately    following the vector's coding region for the 6 histidinyl residues.    In the 3′-end, all inserted gene fragments include a stop codon.-   2. The vectors from 1 are transfected into the E. Coli M15[pREP4]    strain, which contains an expression as well as a repressor plasmid    facilitating proper expression.-   3. The vectors from 1 are further inserted into the E. coli XL1 Blue    for long-time storage.-   4. The transfected and selected clones are tested for expression in    small scale whereby optimum conditions for expression in terms of    the amount of IPTG, the density of cells and the time of expression    induction are determined.-   5. From the information obtained in 4, large scale cultures are    established; subsequently the expression products are harvested and    purified on a Ni-NTA column.-   6. Purity and yield of the large-scale expression is investigated by    means of SDS-PAGE and spectrophotometry, whereafter the proteins are    aliquoted for use in immunization experiments and other experiments.    Immunization and S. aureus Challenge Infection in Mice (Zhou et al.    2006 Vaccine 24. 4830-4837)-   1. 2 months old NMRI mice were used.-   2. Groups of 8 mice (unless other numbers are indicated) were used    for immunization. The mice were immunized 3 times (at day 0, 14,    and 28) prior to challenge infection. A control group of 8 mice was    treated according to an identical protocol with the exception that    an irrelevant protein antigen was used for immunization.    1st Immunization:

50 μg protein (per mice) was mixed with 100 μl aluminum hydroxide(Alhydrogel 2.0%, Brenntag) per 125 μg protein and incubated withend-over-end rotation for 15 min. Freund's incomplete adjuvant (sigma)was added in the volume 1:1 and the mixture was vortexed thoroughly for1 hour. This mixture was injected subcutaneously

2nd and 3rd Immunization

The mice were booster injected intraperitoneally with 2 weeks interval,using the same amount of protein mixed with aluminum hydroxide andphysiological saline solution.

-   3. One week after the last immunization 250 μl blood is drawn from    the mice in order to determine the antibody titer.-   4. 14 days after the last immunization, a number of bacteria (2×10⁹    cells) corresponding to a predetermined LD₉₀ in the control group of    mice was administered intraperitoneally to all mice.

The cells were handled cold and kept on ice until use. The stocksolution of MRSA cells were thawed on ice and then the appropriateamount of cells in sterile physiological saline (total volume per mouse500 μl).

The survival was surveilled twice daily in the first 48 hours afterchallenge and once daily in the subsequent 7 days. The mice weresacrificed if they showed signs of suffering. The mice were monitoredwith respect to loss of weight and body temperature using an implantedchip. The organs of the mice were used for determination of CFU counts.

Test of Antibody Titer

Maxisorp microtiter plates (Nunc, Roskilde, Denmark) were coatedovernight with 1 μg/ml recombinant peptide (His-tagged SAR-protein), 100μl/well.

The next day the plates were emptied and washed 3 times in PBS-Tw. Afterthe last wash the plates were allowed to stand in PBS-Tw for a minimumof 15 minutes (blocking step).

EDTA plasma was diluted 1:100 in PBS-Tw and 200 μl was added to thefirst well. 100 μl of washing buffer was added to all of the otherwells.

A 2-fold dilution was made by transferring 100 ml from the first well tothe next, and so on. The plates were incubated at room temperature for 2hours with shaking. The plates were washed and 100 μl of secondaryantibody was added per well (e.g. HRP conjugated polyclonal rabbitanti-mouse immunoglobulin) and then incubated for 1 hour at roomtemperature with shaking.

The plates were washed and the ELISA developed.

The optical density value was used to calculate the antibody titer:[1/Dilution at ½ max absorbance].

Buffers used were:

-   Coating buffer: 15 mM Na₂CO₃, 35 mM NaHCO₃-   PBS-Tw: PBS, 0.05% Tween-20 pH 7.4-   Coloring buffer for developing the ELISA: 7.3 g citric acid, 11.86 g    NaHPO₄ pH 5.0 at 1 L. OPD tablets (KemEnTec, Diagnostic) were added,    2 mg per 5 ml coloring buffer. Immediately before use, 2 ml of 35%    H₂O₂ was added per tablet. 100 ml of the mixed coloring substrate    was added to each well. The reaction was stopped with 100 ml 1M    H₂SO₄.    Result of Challenge Studies

The polypeptides used in the challenge studies described are in thefollowing section setting forth the results provided with identificationnumbers in the format “SARXXXX”. For easy reference, these polypeptidesrelate to the SEQ ID NOs used herein according to the following table:

SEQ ID 1: SAR2104 SEQ ID 2: SAR1879 SEQ ID 3: SAR0730 SEQ ID 4: SAR2722SEQ ID 5: SAR1507 SEQ ID 6: SAR0222 SEQ ID 7: SAR1558 SEQ ID 8: SAR1026SEQ ID 9: SAR1489 SEQ ID 10: SAR1819 SEQ ID 11: SAR0826 SEQ ID 12:SAR0390 SEQ ID 13: SAR0280 SEQ ID 14: SAR1816 SEQ ID 15: SAR0992 SEQ ID16: SAR1881 SEQ ID 17: SAR0735 SEQ ID 18: SAR2119 SEQ ID 19: SAR2184

The challenge study gave the following results in term of overallsurvival in the vaccinated groups vs. the control groups:

Percentage of mice surviving at end of experiment Vaccine proteinVaccinated mice Control control group SAR2104-20-154 50%  0%SAR0280-28-820 75%  0% SAR0390-21-190  0%  0% SAR2104-20-154 25%  0%*SAR1879-24-184  14%*  0%* SAR0222-27-609 13%  0%* SAR1881-25-208 13% 0%* SAR2119-34-370 25%  0%* SAR0872-27-273  29%*  0%* SAR2718-24-157 17%**  0%* SAR1816-1-27  67%** 0% (50%) SAR0735-26-227 88% 0% (50%)SAR0992-428-769  29%* 0% (50%) SAR1816-46-396 63% 0% (50%) 1:1:1 Mixtureof  88%^(#)   0%^(#) SAR2104-20-154 SAR0280-28-820 SAR0872-27-273SAR0826-42-209  0% (13%) SAR0992-1-409 100%  (13%) SAR1489-343-486 75%(13%) SAR1507-1-652 88% (13%) SAR1558-21-144 38% (10%) SAR0730-22-129100%* (13%) SAR1819-1-1274 88% (13%) SAR2722-920-948 63% (13%)SAR1972-23-91 50% (13%) SAR2104-20-154_nativ 63% (13%)SAR0280-28-820_nativ 63% (13%) *7 mice in group **6 mice in group ^(#)16mice in group

Percentages in parentheses in control group column indicate survivalrate in control group, where mice received injection with adjuvantmixture. Percentages without parentheses in control group columnindicate survival rate in control group, where mice received salineonly.

Results of ELISA Tests

The tables set forth on the following pages show the OD measurementsand, where applicable, in vaccinated mice from the different treatmentgroups:

Test protein: SAR 2104-20-154 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 Juni 2104 18 3 4 4 4 3.777 3.441 2.735 1.958 1.293 0.7330.428 0.246 0.12 Juni 2104 20 3 4 4 4 4 3.789 3.282 2.436 1.6 0.9790.547 0.262 0.121 Juni 2104 22 3 3.894 3.892 3.897 3.871 3.544 3.1222.573 2.006 1.383 0.841 0.481 0.252 Juni 2104 24 3 4 4 4 4 3.719 3.0252.07 1.357 0.825 0.487 0.265 0.138 Juni 2104 25 3 4 4 4 3.86 3.453 2.8242.089 1.497 0.896 0.516 0.29 0.156 Juni 2104 27 3 4 4 3.875 3.667 3.1812.445 1.644 1.037 0.598 0.332 0.175 0.095 Juni 2104 29 3 4 4 4 4 3.5882.656 1.765 1.049 0.588 0.319 0.176 0.089 Juni 2104 31 3 4 4 3.894 3.6893.043 2.225 1.52 1.034 0.614 0.347 0.193 0.101

Test protein: SAR 0280-28-820 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 juni 280 60 3 4 4 4 3.777 3.441 2.735 1.958 1.293 0.7330.428 0.246 0.12 juni 280 61 3 4 4 4 4 3.789 3.282 2.436 1.6 0.979 0.5470.262 0.121 juni 280 62 3 3.894 3.892 3.897 3.871 3.544 3.122 2.5732.006 1.383 0.841 0.481 0.252 juni 280 63 3 4 4 4 4 3.719 3.025 2.071.357 0.825 0.487 0.265 0.138 juni 280 64 3 4 4 4 3.86 3.453 2.824 2.0891.497 0.896 0.516 0.29 0.156 juni 280 65 3 4 4 3.875 3.667 3.181 2.4451.644 1.037 0.598 0.332 0.175 0.095 juni 280 66 3 4 4 4 4 3.588 2.6561.765 1.049 0.588 0.319 0.176 0.089 juni 280 67 3 4 4 3.894 3.689 3.0432.225 1.52 1.034 0.614 0.347 0.193 0.101

Test protein: SAR 0390-21-190 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 juni 0390* 50 3 4 4 4 4 3.593 2.83 1.979 1.253 0.714 0.3890.208 0.121 juni 0390** 51 3 4 4 4 3.925 3.627 2.731 1.785 1.053 0.5740.296 0.155 0.081 juni 0390 52 3 4 4 4 4 3.516 2.709 1.86 1.11 0.6270.333 0.182 0.095 juni 0390 53 3 4 4 4 4 3.753 3.297 2.379 1.538 0.9170.502 0.278 0.15 juni 0390 54 3 4 4 4 4 3.865 3.446 2.648 1.799 1.0770.6 0.337 0.163 juni 0390 55 3 4 4 4 4 3.931 3.907 3.544 2.769 1.8441.107 0.612 0.316 juni 0390 56 3 4 4 4 3.864 3.396 2.59 1.715 1.0430.584 0.317 0.173 0.093 juni 0390 57 3 4 3.931 4 3.841 3.233 2.309 1.4430.83 0.45 0.234 0.123 0.067

Test protein: SAR0222-27-609 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 222 0Ø 3 3.899 3.646 3.306 2.755 2.035 1.351 0.806 0.4850.289 0.177 0.098 0.06 222 BØ 3 3.011 2.854 2.518 1.964 1.485 0.9690.607 0.375 0.243 0.128 0.077 0.04 222 VØ 3 3.669 3.294 2.737 1.98 1.3140.784 0.451 0.268 0.175 0.098 0.063 0.034 222 HØ 3 3.535 3.375 3.242.814 2.317 1.824 1.466 1.166 0.889 0.576 0.344 0.185 222 20Ø 3 3.0552.927 2.803 2.498 1.949 1.513 1.087 0.756 0.481 0.283 0.157 0.086 2222BØ 3 2.568 2.281 1.871 1.331 0.84 0.471 0.267 0.145 0.084 0.049 0.0290.017 222 2HØ 3 4 3.816 3.646 3.093 2.389 1.66 1.033 0.628 0.415 0.250.143 0.077 222 2VØ 3 4 3.827 3.552 3.05 2.385 1.665 1.065 0.642 0.3650.203 0.127 0.062

Test protein: SAR0872-27-273 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 0872 2 3 4 4 4 4 4 4 4 4 4 3.564 2.528 1.57 0872 4 3 4 4 44 4 4 4 3.944 3.476 2.441 1.561 0.917 0872 6 3 4 4 4 4 4 4 4 4 3.9313.367 2.446 1.57 0872 8 3 4 4 4 4 4 4 4 4 4 3.777 2.732 1.739 0872 17 34 4 4 4 4 4 4 4 3.724 2.838 1.913 1.158 0872 19 3 4 4 4 4 4 4 4 3.7863.121 2.237 1.409 0.811 0872 21 3 4 4 4 4 4 4 4 4 3.566 2.552 1.6150.901 0872 23 3 4 4 4 4 4 4 4 4 3.887 3.269 2.251 1.404

Test protein: SAR1879-24-184 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 1879 0Ø 3 4 4 4 4 4 4 4 3.871 3.028 2.038 1.228 0.662 1879VØ 3 4 4 4 4 4 4 3.508 2.428 1.548 0.881 0.478 0.242 1879 HØ 3 4 4 4 4 44 3.665 2.686 1.778 1.041 0.577 0.301 1879 20Ø 3 4 4 4 4 4 4 3.501 2.4681.548 0.856 0.474 0.244 1879 2BØ 3 4 4 4 4 4 3.872 3.175 2.12 1.3330.747 0.406 0.213 1879 2VØ 3 4 4 4 4 4 4 4 3.52 2.453 1.483 0.821 0.4281879 2HØ 3 4 4 4 4 4 3.885 3.363 2.325 1.452 0.826 0.448 0.224

Test protein: SAR1881-25-208 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 1881 0Ø 3 4 4 3.66 3.06 2.238 1.454 0.848 0.47 0.251 0.1350.078 0.047 1881 BØ 3 4 4 3.558 2.546 1.551 0.884 0.481 0.26 0.137 0.0750.048 0.032 1881 VØ 3 4 4 3.78 2.952 1.888 1.105 0.617 0.335 0.181 0.1020.058 0.039 1881 HØ 3 4 3.595 2.644 1.678 0.983 0.546 0.3 0.162 0.0910.053 0.035 0.026 1881 20Ø 3 4 4 4 4 3.583 3.018 2.231 1.454 0.866 0.4780.258 0.141 1881 2BØ 3 1.195 0.577 0.316 0.171 0.101 0.064 0.044 0.0320.027 0.022 0.022 0.019 1881 2VØ 3 4 3.792 3.313 2.508 1.716 1.024 0.5980.333 0.181 0.1 0.06 0.039 1881 2HØ 3 0.995 0.559 0.3 0.165 0.097 0.0610.043 0.033 0.026 0.02 0.019 0.02

Test protein: SAR2104-20-154 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 2104 0Ø 3 4 4 4 4 4 4 3.841 3.033 2.031 1.197 0.674 0.3712104 BØ 3 0.67 0.377 0.22 0.138 0.089 0.067 0.052 0.048 0.044 0.0420.041 0.04 2104 VØ 3 0.276 0.165 0.099 0.073 0.057 0.053 0.047 0.0450.043 0.042 0.041 0.039 2104 20Ø 3 0.153 0.099 0.071 0.057 0.048 0.0470.043 0.043 0.044 0.042 0.041 0.039 2104 2BØ 3 0.458 0.378 0.285 0.2440.2 0.169 0.135 0.099 0.07 0.052 0.047 0.043 2104 2VØ 3 0.349 0.25 0.1780.133 0.098 0.09 0.067 0.041 0.046 0.043 0.042 0.039 2104 2HØ 3 0.3450.221 0.142 0.103 0.074 0.06 0.051 0.047 0.044 0.041 0.042 0.037

Test protein: SAR2119-34-370 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 2119 0Ø 3 4 4 4 3.857 4 4 3.689 3.27 1.839 1.121 0.5970.311 2119 BØ 3 4 4 4 4 4 4 3.872 3.379 2.373 1.49 0.827 0.434 2119 VØ 34 4 3.949 4 4 4 3.892 3.218 2.22 1.339 0.751 0.381 2119 HØ 3 0.187 0.1010.168 0.102 0.062 0.041 0.03 0.022 0.017 0.017 0.015 0.011 2119 20Ø 3 44 4 4 3.421 2.446 1.457 0.819 0.439 0.228 0.122 0.063 2119 2BØ 3 4 4 4 44 4 4 3.594 2.695 1.747 1.026 0.547 2119 2VØ 3 4 4 4 4 3.577 2.731 1.7341.012 0.552 0.285 0.146 0.076 2119 2HØ 3 4 4 4 4 3.498 2.52 1.521 0.8740.466 0.244 0.128 0.066

Test protein: SAR2718-24-157 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 2718 0Ø 3 4 4 4 4 4 3.906 3.27 2.491 1.673 1.062 0.6160.323 2718 VØ 3 3.923 4 4 3.786 3.09 2.101 1.238 0.682 0.366 0.19 0.1050.057 2718 HØ 3 4 4 4 4 4 3.944 3.498 2.547 1.622 0.963 0.514 0.27 271820Ø 3 4 4 4 4 3.944 2.971 1.885 0.966 0.449 0.187 0.086 0.047 2718 2BØ 34 4 3.885 3.344 3.204 1.371 0.773 0.402 0.216 0.115 0.06 0.037 2718 2VØ3 4 4 4 4 3.801 2.893 1.911 1.17 0.632 0.349 0.174 0.091 2718 2HØ 3 4 44 3.726 3.229 1.844 1.104 0.595 0.324 0.174 0.093 0.051

Test protein: SAR0826-42-209 ELISA Mouse Bleed Sera dilution (OD490 nm)Plate ID No Bleed # 100 200 400 800 1600 3200 6400 12800 25600 51200102400 204800 0826 1 3 −0.0058 −0.0288 −0.0138 −0.0258 −0.0128 −0.02880.0132 0.0062 −0.0688 −0.0098 −0.0398 −0.0178 0826 2 3 0.2672 −0.0188−0.0668 −0.0888 −0.1188 −0.1038 −0.1288 −0.1168 −0.1188 −0.1068 −0.06980.3492 0826 3 3 −0.0828 −0.1518 −0.1278 −0.1308 −0.1268 −0.1168 −0.1288−0.1338 −0.0558 −0.1118 −0.0968 −0.0378 0826 4 3 −0.0918 −0.1458 −0.1308−0.1448 −0.1408 −0.1318 −0.1518 −0.1398 −0.1328 −0.1168 −0.1068 −0.05880826 5 3 0.0922 −0.1228 −0.1448 −0.1438 −0.1168 −0.1198 −0.0918 −0.1388−0.1288 −0.1138 −0.1228 −0.0908 0826 6 3 1.1902 0.4132 0.0792 −0.0518−0.1048 −0.1448 −0.1378 −0.1568 −0.1378 −0.1168 −0.1168 −0.0678 0826 7 33.1572 2.6172 1.6322 0.8702 0.3652 0.1352 −0.0218 −0.0928 −0.0928−0.0988 −0.0948 −0.0718 0826 8 3 −0.1568 −0.1638 −0.1168 −0.1198 −0.02380.0102 −0.0388 −0.0828 −0.0818 −0.0618 −0.0368 −0.0038Immune Fluorescence/FACS Analyses of Plasma Samples from Immunized Mice

-   1. Groups of mice will be immunized three times with intervals of 14    days with antigen coupled onto carrier-proteins, diphtheria-toxoid    and/or secreted mycobacterial proteins (PPD). All immunizations are    carried out subcutaneously with the antigen adsorbed onto Al(OH)₃    and with Freunds incomplete adjuvant, cf. above.-   2. A control group of mice are immunized with diphtheria-toxoid    without antigen.-   3. Mice are bled after the second and the third immunization.-   4. The serum bleeds are tested for their reactivity against the    immunizing antigen.-   5. The following methods will be used:    -   Direct measurement of antibodies to the immunizing antigen    -   Analysis for agglutinating power when antibodies are incubated        with the bacteria    -   Analysis for killing effect on bacteria after incubation of        bacteria with antiserum+fresh serum (complement)        Proteins of the Invention

The S. aureus proteins of the present invention are set forth in thesequence listing together with their related nucleic acid sequences. Foreasy reference, the one letter amino acid sequences of the S. aureusproteins are provided in the following:

SEQ ID NO: 1: MKRLLGLLLVSTLVLSACGNDENQEESKKEVKSKEKKIEKEKENKSKKDKEKEVATQQQPDNQTVEQPQSQEQSVQQPQQQIPQNSVPQQNVQVQQNKKQKVDLNNMPPTDFSTEGMSEQAQKQIEELSMQKDYHGLSQREYNDRVSEII NNDN SEQ ID NO: 2:MLKGCGGCLISFIILIILLSACSMMFSNNDNSTSNQSSKTQLTQKDEDKSENMPEEKSESETDKDLQSTEEVPANENTENNQHEIDEITTTDQSDDEINTPNVAEEESQDDLKDDLKEKQQPSDHHQSTQPKTSPSTETNKQQSFANCKQLRQVYPNGVTADHPAYRPHLDRDKDKRACEPDKY SEQ ID NO: 3:MKKLIISIMAIMLFLTGCGKSQEKATLEKDIDNLQKENKELKDKKEKLQQEKEKLADKQKDLEKEVKDLKPSKEDNKDDKKDEDKNKDKDKEASQDKQSKDQTKSSDKDNHKKPTSTDKDQKANDKHQS SEQ ID NO: 4:MKNAFKLFKMDLKKVAKTPAVWIILAGLAILPSFYAWFNLWAMWDPYGNTGHIKVAVVNEDKGDTIRGKKVNVGNTMVNTLKKNKSFDWQFVSREKADHEIKMGKYFAGIYIPSKFTHEITGTLRKQPQKADVEFKVNQKINAVASKLTDTGSSVWEKANEQFNKTVTRALLEEANKAGLTIEENVPTINKIKNAVYSADKALPKINDFANKIVYLNNHQADLDKYANDFRKLGNYKGDILDAQKKLNEVNGAIPQLNEKAKULALNNYMPKIEKALNFAADDVPAQFPKINQGLNIASQGIDQANGQLNDAKGFVTQVRSRVGDYQEAIRRAQDLNRRNQQQIPQNSAANNETSNSAPAAGNGVTSTPPSAPNGNTTPNNNVTQNTAPNSNNAPVSTTPQSTSGKKDGQSFADnTTQVSTANENTQNITDKDVKSMEAALTGSLLSLSNNLDTQAKAAQKDSQALRNISYGILASDKPSDFRESLDNVKSGLEYTTQYNQQFIDTLKEIEKNENVDLSKEIDKVKTANNRINESLRLVNQLSNALKNGSSGTAEATKLLDQLSKLDSSLSSFRDYVKKDLNSSLVSISQRIMDELNKGQTALSNVQSKLNTIDQVINSGQSILKNGKTRIDRLQTVLPSIEQQYISAIKNAQANFPKVKSDVAKAANFVRNDLPQLEQRLTNATASVNKNLPTLLNGYDQAVGLLNKNQPQAKKALSDLADFAQNKLPDVEKDLKKANKIFKKLDKDDAVDKLIDTLKNDLKKQAGIIANPINKKTVDVFPVKDYGSGMTPFYTALSWVGALLMVSLLTVDNKHKSLEPVLTTRQVFLGKAGFFIMLGMLQAUVSVGDLLILKAGVESPVLFVLITIFCSIIFNSIVYTCVSLLGNPGKAIAIVLLVLQIAGGGGTFPIQTTPQFFQNISPYLPFTYAIDSLRETVGGIVPEILITKLIILTLFGIGFFWGLILKPVTDPLMKRVSEKVDQSNVTE SEQ ID NO: 5:MNEKVEGMTLELKLDHLGVQEGMKGLKRQLGVVNSEMKANLSAFDKSEKSMEKYQARIKGLNDRLKVQKKMYSQVEDELKQVNANYQKAKSSVKDVEKAYLKLVEANKKEKLALDKSKEALKSSNTELKKAENQYKRTNQRKQDAYQKLKQLRDAEQKLKNSNQATTAQLKRASDAVQKQSAKHKALVEQYKQEGNQVQKLKVQNDNLSKSNDKIESSYAKTNTKLKQTEKEFNDLNNTIKNHSANVAKAETAVNKEKAALNNLERSIDKASSEMKTFNKEQMIAQSHFGKLASQADVMSKKFSSIGDKMTSLGRTMTMGVSTPITLGLGAALKTSADFEGQMSRVGAIAQASSKDLKSMSNQAVDLGAKTSKSANEVAKGMEELAALGFNAKQTMEAMPGVISAAEASGAEMATTATVMASAINSFGLKASDANHVADLLARSANDSAADIQYMGDALKYAGTPAKALGVSIEDTSAAIEVLSNSGLEGSQAGTALRASFIRLANPSKNTAKEMKKLGIHLSDAKGQFVGMGELIRQFQDNMKGMTREQKLATVATIVGTEAASGFLALIEAGPDKINSYSKSLKNSNGESKKAADLMKDNLKGALEQLGGAFESLAIEVGKDLTPMIRAGAEGLTKLVDGFTHLPGWVRKASVGLALFGASIGPAVLAGGLLIRAVGSAAKGYASLNRRIAENTILSNTNSKAMKSLGLQTLFLGSTTGKTSKGFKGLAGAMLFNLKPINVLKNSAKLAILPFKLLKNGLGLAAKSLFAVSGGARFAGVALKFLTGPIGATITAITIAYKVFKTAYDRVEWFRNGINGLGETIKFFGGKIIGGAVRKLGEFKNYLGSIGKSFKEKFSKDMKDGYKSLSDDDLLKVGVNKFKGFMQTMGTASKKASDTVKVLGKGVSKETEKALEKYVHYSEENNRIMEKVRLNSGQITEDKAKKLLKIEADLSNNLIAEIEKRNKKELEKTQELIDKYSAFDEQEKQNILTRTKEKNDLRIKKEQELNQKIKELKEKALSDGQISENERKEIEKLENQRRDITVKELSKTEKEQERILVRMQRNRNSYSIDEASKAIKEAEKARKAKKKEVDKQYEDDVIAIKNNVNLSKSEKDKLLAIADQRHKDEVRKAKSKKDAWDWKKQNKDIDKEMDLSSGRVYKNTEKWWNGLKSWWSNFREDQKKKSDKYAKEQEETARRNRENIKKWFGNAWDGVKSKTGEAFSKMGRNANHFGGEMKKMWSGIKGIPSKLSSGWSSAKSSVGYHTKAIANSTGKWFGKAWQSVKSTTGSIYNQTKQKYSDASDKAWAHSKSIWKGTSKWFSNAYKSAKGWLTDMANKSRSKWDNISSTAWSNAKSVWKGTSKWFSNSYKSLKGWTGDMYSRAHDRFDAISSSAWSNAKSVFNGFRKWLSRTYEWIRDIGKDMGRAAADLGKNVANKAIGGLNSMIGGINKISKAITDKNLIKPIPTLSTGTLAGKGVATDNSGALTQPTFAVLNDRGSGNAPGGGVQEVIHRADGTFHAPQGRDWVPLGVGDSVINANDTLKLQRMGVLPKFHGGTKKKKWMEQVTENLGKKAGDFGSKAKNTAHNIKKGAEEMVEAAGDKIKDGASWLGDKIGDVWDYVQHPGKLVNKVMSGLNINFGGGAIMATVKIAKGAYSLLKKKLVDKVKSWFEDFGGGGDGSYLFDHPIWQRFGSYTGGLNFNGGRHYGIDFQMPTGTNIYAVKGGIADKVWTDYGGGNSIQIKTGANEWNWYMHLSKQLARQGQRIKAGQLIGKSGATGNFVRGAHLHFQLMQGSHPGNDTAKDPEKWLKSLKGSGVRSGSGVNKAASAWAGDIRRAAKRMGVNVTSGDVGNIISUQHESGGNAGITQSSALRDINVLQGNPAKGLLQYIPQTFRHYAVRGHNNIYSGYDQLLAFFNNSYWRSQFNPRGGWSPSGPRRYANGGLITKHQLAEVGEGDKQEMVIPLTRRKRAIQLTEQVMRIIGMDGKPNNITVNNDTSTVEKLLKQIVMLSDKGNKLTDALIQTVSSQDNNLGSNDAIRGLEKILSKQSGH RANANNYMGGLTNSEQ ID NO: 6: MKKQIISLGALAVASSLFTWDNKADAIVTKDYSKESRVNENSKYDTPIPDWYLGSILNRLGDQIYYAKELTNKYEYGEKEYKQAIDKLMTRVLGEDHYLLEKKKAQYEAYKKWFEKHKSENPHSSLKKIKFDDFDLYRLTKKEYNELHQSLKEAVDEFNSEVKNIQSKQKDLLPYDEATENRVTNGIYDFVCEIDTLYAAYFNHSQYGHNAKELRAKLDIILGDAKDPVRITNERIRKEMMDDLNSIIDDFFMDTNMNRPLNITKFNPNIHDYTNKPENRDNFDKLVKETREAIANADESWKTRTVKNYGESETKSPWKEEKKVEEPQLPKVGNQQEDKITVGTTEEAPLPIAQPLVKIPQGTIQGEIVKGPEYLTMENKTLQGEIVQGPDFPTMEQNRPSLSDNYTQPTTPNPILKGIEGNSTKLEIKPQGTESTLKGTQGESSDIEVKPQATETTEASHYPARPQFNKTPKYVKYRDAGTGIREYNDGTFGYEARPRFNKPSETNAYNVTTNQDGTVSYGARPTQNKPSETNAYNVTTHANGQVSYGARPTQNKPSETNAYNVTTHANGQVSYGARPTQNKPSKTNAYNVTTHADGTA TYGPRVTKSEQ ID NO: 7: MKKVIGLLLVSTLALTACGEKEKPKKEENKKSQTQKHKDSKPKTQQEKMKKVEDKNPPNNSIQNNSNNQNQSQNNQLNNNSDPSNNTPANINKNDSQNTNLNDEYWSPGWTKDEQAKAFEEYKKGKEDEARAGASAVPGANIN SEQ ID NO: 8:MAKKFNYKLPSMVALTLVGSAVTAHQVQAAETTQDQTTNKNVLDSNKVKATTEQAKAEVKNPTQNISGTQWQDPAIVQPKAANKTGNAQVNQKVDTTQVNGDTRATQSTTSNNAKPVTKSTNTTAPKTNNNVTSAGYSLVDDEDDNSENQINPELIKSAAKPAALETQYKAAAPKATPVAPKAKTEATPKVTTFSASAQPRSAAAAPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIWHDTANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASFARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDLINEKYLIKMGKVAPWGTQSTTTPTTPSKPSTPSKPSTPSTGKLTVAANNGVAQIKPTNSGLYTTVYDKTGKATNEVQKTFAVSKTATLGNQKFYLVQDYNSGNKFGWVKEGDWYNTAKSPVNVNQSYSIKPGTKLYTVPWGTSKQVAGSVSGSGNQTFKASKQQQIDKSIYLYGSVNGKSGWVSKAYLVDTAKPTPTPTPKPSTPTTNNKLTVSSLNGVAQINAKNNGLFTTVYDKTGKPTKEVQKTFAVTKEASLGGNKFYLVKDYNSFTLIGWVKQGDVIYNNAKSPVNVMQTYTVKPGTKLYSVPWGTYKQEAGAVSGTGNQTFKATKQQQIDKSIYLYGTVNGKSGWISKAYLAVPAAPKKAVAQPKTAVKAYAVTKPQTTQTVSKIAQVKPNNTGIRASVYEKTAKNGAKYADRTFYVTKERAHGNETYVLLNNTSHNIPLGWFNVKDLNVQNLGKEVKTTQKYTVNRSNNGLSMVPWGTKNQVILTGNNIAQGTFNATKQVSVGKDVYLYGTINNRTGWVNSKDLTAPTAVKPTTSAAKDYNYTYVIKNGNGYYYVTPNSDTAKYSLKAFNEQPFAWKEQVINGQTWYYGKLSNGKLAWIKSTDLAKELIKYNQIGMTLNQVAQIQAGLQYKPQVQRVPGKWTDANFNDVKHAMDTKRLAQDPALKYQFLRLDQPQNISIDKINQFLKGKGVLENQGAAFNKAAQMYGINEVYLISHALLETGNGTSQLAKGADWNNKWTNSNTKYHNVFGIAAYDNDPLREGIKYAKQAGWDTVSKAIVGGAKFIGNSYVKAGQNTLYKMRWNPAHPGTHQYATDVDWANINAKIIKGYYDKIGEVGKYFDIPQYK SEQ ID NO: 9:MSNNFKDDFEKNRQSIDTNSHQDHTEDVEKDQSELEHQDTIENTEQQFPPRNAQRRKRRRDLATNHNKQVHNESQTSEDNVQNEAGTIDDRQVESSHSTESQEPSHQDSTPQHEEGYYNKNAFAMDKSHPEPIEDNDKHETIKEAENNTEHSTVSDKSEAEQSQQPKPYFATGANQANTSKDKHDDVTVKQDKDESKDHHSGKKGAAIGAGTAGVAGAAGAMGVSKAKKHSNDAQNKSNSGKVNNSTEDKASEDKSKEHHNGKKGAAIGAGTAGLAGGAASNSASAASKPHASNNASQNNDEHDHHDRDKERKKGGMAKVLLPLIAAVLIIGALAIFGGMALNNHNNGTKENKIANTNKNNADESKDKDTSKDASKDKSKSTDSDKSKDDQDKATKDESDNDQNNANQANNQAQNNQNQQQANQNQQQQQQRQGGGQRHTVNGQENLYRIAIQYYGSGSPENVEKIRRANGLSGNNIRNGQQIVIP SEQ ID NO: 10:MSWFDKLFGEDNDSNDDLIHRKKKRRQESQNIDNDHDSLLPQNNDIYSRPRGKFRFPMSVAYENENVEQSADTISDEKEQYHRDYRKQSHDSRSQKRHRRRRNQTTEEQNYSEQRGNSKISQQSIKYKDHSHYHTNKPGTYVSAINGIEKETHKSKTHNIYSNNTNHRAKDSTTDYHKESFKTSEVPSAIFGTMKPKKLENGRIPVSKSSEKVESDKQKYDKYVAKTQTSQNKHLEQEKQKDSWKQGTASKSSDENVSSTTKSTPNYSKVDNTIKIENIYASQIVEEIRRERERKVLQKRRFKKALQQKREEHKNEEQDAIQRAIDEMYAKQAERYVGDSSLNDDSDLTDNSTEASQLHTNEIEDEAVSNDENKKASIQNEDTDDTHVDESPYNYEEVSLNQVSTTKQLSDDEVTVSDVTSQRQSALQHNVEVNNQDELKNQSRLIADSEEDGATNEEEYSGSQIDDAEFYELNDTEVDEDTTSNSEDNTNRDASEMHVDAPKTQEHAVTESQVNNIDKTVDNEIELAPRHKKDDQTNLSVNSLKTNDVNDGHWEDSSMNEIEKQNAEITENVQNEAAESKQNVEEKTIENVNPKKQTEKVSTLSKRPFNVVMTPSDKKRMMDRKKHSKVNVPELKPVQSKQAASESKTATQNTPSSSTDSQESNTNAYKTNNMTSNNVENNQLIGHAATENDYQNAQQYSEQKPSADSTQTEIFEESQDDNQLENEQVDQSTSSSVSEVSDITEESEETTHQNNTSGQQDNDDQQKDLQLSFSNQNEDTANENRPRTNQPDVATNQAVQTSKPMIRKGPNIKLPSVSLLEEPQVIEPDEDWITDKKKELNDALFYFNVPAEVQDVTEGPSVTRFELSVEKGVKVSRITALQDDIKMALAAKDIRIEAPIPGTSRVGIEVPNQNPTTVNLRSIIESPSFKNAESKLTVAMGYRINNEPLLMDIAKTPHALIAGATGSGKSVCINSILMSLLYKNHPEELRLLLIDPKMVELAPYNGLPHLVAPVITDVKAATQSLKWAVEEMERRYKLFAHYHVRNITAFNKKAPYDERMPKIVIVIDELADLMMMAPQEVEQSIARIAQKARACGIHMLVATQRPSVNVITGLIKANIPTRIAFMVSSSVDSRTILDSGGAERLLGYGDMLYLGSGMNKPIRVQGTFVSDDEIDDVVDFIKQQREPDYLFEEKELLKKTQTQSQDELFDDVCAFMVNEGHISTSLIQRHFQIGYNRAARIIDQLEQLGYVSSANGSKPRDVYVTEADLNKE SEQ ID NO: 11:MSNQNYDYNKNEDGSKKKMSTTAKVVSIATVLLLLGGLVFAIFAYVDHSNKAKERMLNEQKQEQKEKRQKENAEKERKKKQQEEKEQNELDSQANQYQQLPQQNQYQYVPPQQQAPTKQRPAKEENDDKASKDESKDKDDKASQDKSDDNQKKTDDNKQPAQPKPQPQQPTPKPNNNQQNNQSNQQAKPQAPQQNSQSTT NKQNNANDKSEQ ID NO: 12: MKLKSLAVLSMSAVVLTACGNDTPKDETKSTESNTNQDTNTTKDVIALKDVKTSPEDAVKKAEETYKGQKLKGISFENSNGEWAYKVTQQKSGEESEVLVDDKNKKVINKKTEKEDTVNENDNFKYSDAIDYKKAIKEGQKEFDGDIKEWSLEKDDGKLVYNIDLKKGNKKQEVTVDAKNGKVLKSEQDQ SEQ ID NO: 13:MKKKNWIYALIVTLiniAIVSMIFFVQTKYGDQSEKGSQSVSNKNNKIHIAIVNEDQPTTYNGKKVELGQAFIKRLANEKNYKFETVTRNVAESGLKNGGYQVMIVIPENFSKLAMQLDAKTPSKISLQYKTAVGQKEEVAKNTEKWSNVLNDFNKNLVEIYLTSIIDNLHNAQKNVGAIMTREHGVNSKFSNYLLNPINDFPELFTDTLVNSISANKDITKWFQTYNKSLLSANSDTFRVNTDYNVSTLIEKQNSLFDEHNTAMDKMLQDYKSQKDSVELDNYINALKQMDSQIDQQSSMQDTGKEEYKQTVKENLDKLREIIQSQESPFSKGMIEDYRKQLTESLQDELANNKDLQDALNSIKMNNAQFAENLEKQLHDDIVKEPDSDTTFIYNMSKQDFIAAGLNEDEANKYEAIVKEAKRYKNEYNLKKPLAEHINLTDYDNQVAQDTSSLINDGVKVQRTETIKSNDINQLTVATDPHFNFEGDIKINGKKYDIKDQSVQLDTSNKEYKVEVNGVAKLKKDAEKDFLKDKTMHLQLLFGQANRQDEPNDKKATSVVDVTLNHNLDGRLSKDALSQQLSALSRFDAHYKMYTDTKGREDKPFDNKRLIDMMVDQVINDMESFKDDKVAVLHQIDSMEENSDKLIDDILNNKKNTTKNKEDISKLIDQLENVKKTFAEEPQEPKIDKGKNDEFNTMSSNLDKEISRISEKSTQLLSDTQESKTIADSVSGQLNQLDNNVNKLHATGRALGVRANDLNRQMAKNDKDNELFAKEFKKVLQNSKDGDRQNQALKAFMSNPVQKKNLENVLANNGNTDVISPTLFVLLMYLLSMITAYIFYSYERAKGQMNFIKDDYSSKNNLWNNAITSGVIGATGLVEGLIVGLIAMNKFHVLAGYRAKFILMVILTMMVFVLINTYLLRQVKSIGMFLMIAALGLYFVAMNNLKAAGQGVTNKISPLSYIDNMFFNYLNAEHPIGLALVILTVLVIIGFVLNMFIKH FKKERLISEQ ID NO: 14: MTQQQNNKRTLKNKHTYQNEPLPNRKDFVVSFITGALVGSALGLYFKNKVYQKADDLKVKEQELSQKFEERKTQLEETVAFTKERVEGFLNKSKNEQAALKAQQAAIKEEASANNLSDTSQEAQEIQEAKREAQTETDKSAAVSNEESKASALKAQQAAIKEEASANNLSDTSQEAQAIQEVKKEAQAETDKSADVSNEESKASTLNVSKEESQAERLANAAKQKQAKLTPGSKESQLTEALFAEKPVAKNDLKEIPLLVTKKNDVSETVNTDNKDTVKQKEAKFENGVITRKADEKTPNNTAVDKKSGKQSKKTTPSNKRNASKASTNKTSGQKKQHNKKASQGAKKQSSSSNSTTKTNQKNSKATNAKSSNASKKSNAKVEKAKSKIEKRTFND SEQ ID NO: 15:MDIGKKHVIPKSQYRRKRREFFHNEDREENLNQHQDKQNIDNTTSKKADKQIHKDSIDKHERFKNSLSSHLEQRNRDVNENKAEESKSNQGSKSAYNKDHYLTDDVSKKQNSLDSVDQDTEKSKYYEQNTEATLSTNSTDKVESTDMRKLSSDKNKVGHEEQHVLSKPSEHDKETRIDFESSRTDSDSSMQTEKIKKDSSDGNKSSNLKSEVISDKSNSVPILSESDDEVNNQKPLTLPEEQKLKRQQSQNEQTKTYTYGDSEQNDKSNHENDLSHHTPSISDDKDYVMREDHIVDDNPDNDINTPSLSKIDDDRKLDEKIHVEDKHKQNADSSETVGYQSQSSASHRSTEKRNMAINDHDKLNGQKPNTKTSANNNQKKATSKLNKGRATNNNYSAILKKFWMMYWPKLVILMGIIILIVILNAIFNNVNKNDRMNDNNDADAQKYTTTMKNANNAVKSWTVENETSKDSSLPKDKASQDEVGSGVVYKKSGDTLYIVTNAHWGDKENQKITFSNNKSVVGKVLGKDKWSDLAVVKATSSDSSVKEIAIGDSNNLVLGEPILWGNPLGVDFKGTVTEGIISGLNRNVPIDFDKDNKYOMLMKAFQIDASVNPGNSGGAWNREGKLIGWAAKISMPNVENMSFAIPVNEVQKIVKELETKGKIDYPDVGVKMKNIASLNSFERQAVKLLGKVKNGWVDQVDNNGLADQSGLKKGDVITELDGKLLEDDLRFRQIIFSHKDDLKSITAKIY RDGKEKEINIKLKSEQ ID NO: 16: MKFKAIVAITLSLSLLTACGANQHKENSSKSNDTNKKTQQTDNTTQSNTEKQMTPQEAEDIVRNDYKARGANENQTLNYKTNLERSNEHEYYVEHLVRDAVGTPLKRCAIVNRHNGTIINIFDDMSEKOKEEFEAFKKRSPKYNPGMNDQAEMDNESEDIQHHDIDNNKAIQNDLPDQKVDDKNDKNAVNKEEKHDNREN NSAETKVKSEQ ID NO: 17: MDKKKVIKFMINVLPIVLVPLIVERKRIKQHPDVQKVTDATSKVASKTSAAISNTASDVKEYVGDKKQDFENKRELKKFAREHDPAYIEKKGEKLAKQNRKDADKMNKILQKNIEKRHKEEQKAREKNEIQRIKDMKKSQKYEVKAGLTPNKLDEKTEKKGDKLAEKNRKEIAKMNKKLQKNIEKRHKEEQKRQQEADKARIKSFKKYKDYVAKSASQQNKENNTEA SEQ ID NO: 18:MSYHWFKKMLLSTSMLILSSSSSLGLATHTVEAKDNLNGEKPTTNLNHNVTSPSVNSEMNNNETGTPHESNQAGNEGTGSNSRDANPDSNNVKPDSNNQNPSPDSKPDPNNPNPGPNPKPDPDKPKPNPEPKPDPDKPKPNPDPKPDPDKPKPNPDPKPDPNPNPNPKPDPNKPNPNPSPNPNQPGDSNQSGGSKNGGTWNPNASDGSNQGQWQPNGNQGNSQNPTGNDFVSQRFLALANGAYKYNPYILNQINQLGKEYGEVTDEDIYNIIRKQNFSGNAYLNGLQQQSNYFRFQYFNPLKSERYYRNLDEQVLALITGEIGSMPDLKKPEDKPDSKQRSFEPHEKDDFTVVKKQEDNKKSASTAYSKSWLAIVCSMMVVFSIMLFLFVKRNKKKNKNE SQRR SEQ ID NO: 19:MKKTLLASSLAVGLGIVAGNAGHEAQASEADLNKASLAQMAQSNDQTLNQKPIEAGAYNYTFDYEGFTYHFESDGTHFAWNYHATGANGADMSAQAPATNNVAPSADQSNQVQSQEVEAPQNAQTQQPQASTSNNSQVTATPTESKASEGSSVNVNDHLKQIAQRESGGNIHAVNPTSGAAGKYQFLQSTWDSVAPAKYKGVSPANAPESVQDAAAVKLYNTGGAGHWVTA

The invention claimed is:
 1. A pharmaceutical composition comprising apolypeptide consisting of a) an amino acid sequence consisting of aminoacid residues 28-820 of SEQ ID NO: 13, or b) an amino acid sequencehaving a sequence identity of at least 80% with the amino acid sequenceof a) and having the same length as the amino acid sequence of a), or c)an amino acid sequence consisting of at least 35 contiguous amino acidresidues of 28-820 of SEQ ID NO: 13, or d) an amino acid sequence of a),b) or c), which is fused or conjugated to an immunogenic carriermolecule or a tag, said pharmaceutical composition further comprising animmunological adjuvant and a pharmaceutically acceptable carrier,vehicle or diluent, and said polypeptide being antigenic in a mammal. 2.The pharmaceutical composition according to claim 1, wherein thepolypeptide is capable of inducing, in the mammal, an immune responseagainst multi-resistant S. aureus infection.
 3. The pharmaceuticalcomposition according to claim 1, wherein the immunological adjuvant isselected from an aluminum salt, an oil-in-water emulsion formulation, asaponin adjuvant, a cytokine, Complete Freund's Adjuvant (CFA), andIncomplete Freund's Adjuvant (IFA).
 4. The pharmaceutical compositionaccording to claim 3, wherein the aluminum salt is aluminum hydroxide,aluminum phosphate, or aluminum sulfate.
 5. The pharmaceuticalcomposition according to claim 3, wherein the oil-in-water emulsion isMF59, SAF, or a Ribi adjuvant system.
 6. The pharmaceutical compositionaccording to claim 3, wherein the cytokine is selected from the groupconsisting of an interleukin, an interferon, M-CSF, and TNF.
 7. A unitdose of a pharmaceutical composition comprising a polypeptide consistingof: a) an amino acid sequence consisting of amino acid residues 28-820of SEQ ID NO: 13, or b) an amino acid sequence having a sequenceidentity of at least 80% with the amino acid sequence of a) and hayingthe same length as the amino acid sequence of a), or c) an amino acidsequence consisting of at least 35 contiguous amino acid residues of28-820 SEQ ID NO: 13, or d) an amino acid sequence of a), b) or c),which is fused or conjugated to an immunogenic carrier molecule or a tagwherein the amount of said polypeptide is between 0.5 μg and 500 mg,said pharmaceutical composition further comprising a pharmaceuticallyacceptable carrier, vehicle or diluent, and said polypeptide beingantigenic in a mammal.
 8. The unit dose according to claim 7, whereinthe amount of said polypeptide does not exceed 5,000 μg.
 9. The unitdose according to claim 8, wherein the amount of said polypeptide is inthe range between 10 and 200 μg.
 10. The unit dose according to claim 7,further comprising an immunological adjuvant.
 11. The unit doseaccording to claim 10, wherein the immunological adjuvant is selectedfrom an aluminium salt, an oil-in-water emulsion formulation, a saponinadjuvant, a cytokine, Complete Freund's Adjuvant (CFA), and IncompleteFreund's Adjuvant (IFA).
 12. The unit dose according to claim 11,wherein the aluminium salt is aluminium hydroxide, aluminium phosphate,or aluminium sulfate.
 13. The unit dose according to claim 11, whereinthe oil-in-water emulsion is MF59, SAF, or a Ribi adjuvant system. 14.The unit dose according to claim 11, wherein the cytokine is selectedfrom the group consisting of an interleukin, an interferon, M-CSF, andTNF.